Virus-derived Platforms for Visualizing Protein Associations inside Cells

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 NS 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 NS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of NS 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 NS-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 NS platform in conjunction with threecolor 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. Molecular & Cellular Proteomics 6:1027-1038, 2007.Protein-protein associations are vital to cellular functions. Numerous methods are available for addressing whether proteins associate either directly ("interaction") or indirectly (through bridging molecules), but many of these methods can be technically demanding, can be limited by the strength of the association, or cannot resolve whether the association indeed occurs inside cells. In addition, many current objectives and strategies demand methods that are simple and adaptable to high throughput formats. In this report, we describe a new such method that is based on an unusual property of the orthoreovirus-encoded NS protein.Double-stranded RNA viruses of the taxonomic family Reoviridae (e.g. orbiviruses, orthoreoviruses, and rotaviruses) form characteristically shaped, non-membrane-bound inclusions in the cytoplasm of infected cells. These structures are variously called viral inclusion bodies (orbiviruses) (1), viral factories (orthoreoviruses) (2, 3), or viroplasms (rotaviruses) (4) and are thought to be the sites of viral RNA replication and packaging into assembly intermediates that can later mature into infectious virions. The viral factories of some orthoreoviruses (such as certain isolates of strain Type 3 Dearing (T3D) 1 ), for example, have a globular morphology (5, 6) and can attain large diameters (10 m or more) that make them easily identifiable by light microscopy (see Fig. 1a, left panel).Previous studies have identified the medium-sized (-c...

Localization of Mammalian Orthoreovirus Proteins to Cytoplasmic Factory-Like Structures via Nonoverlapping Regions of μNS

“…pCI-NS(14-721), pCI-NS(41-721), pCI-NS(173-721), pCI-NS(221-721), and pCI-NS(471-721) were described previously but were originally named pCI-M3(14-721), pCI-M3(41-721), pCI-M3(173-721), pCI-M3(221-721), and pCI-M3(471-721), respectively (5,8,33). pCI-3/HA expressing a C-terminally HA-tagged version of 3 was described previously (32). pEGFP/NS(471-721) was described previously but was originally named pEGFP-C1-M3(471-721) (5).…”

“…We previously developed an assay that exploits the characteristic ability of NS, as well as the rotavirus protein NSP5 fused at its amino terminus (N terminus) to enhanced green fluorescent protein (EGFP) (34), to form distinctive structures in the cytoplasm in order to identify and map protein-protein interactions (32). In the current study, we modified and made extensive use of this novel assay to explore further the associations between NS and other MRV proteins.…”

“…These results strongly suggest that the formation of VF is an important and necessary function for successful MRV multiplication. Previous studies have shown that NS associates with six other MRV proteins: five structural proteins that make up the core particle (1, 2, 3, 2, and 2) and the single-stranded RNA (ssRNA) binding nonstructural protein NS (4,6,8,32,33). Limited mapping of NS associations with other viral proteins has shown that NS aa 14 to 41 are necessary and that aa 1 to 41 are sufficient for the association with the minor core protein 2 (8).…”

ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein

“…However, the inactive mutant Figure 6A). In COS-7 cells, which expressed large T antigen known to inhibit the growth-regulatory function of pRB (Gluzman, 1981;Chellappan et al, 1992;Miller et al, 2007), the ARF1 mutant was not found in the nucleus but perinuclearly (Supplementary Figure 6B). To further determine whether the endogenous expression of pRB could regulate the enrichment of endogenous ARF1 or ARF1T 31 N in chromatin, we next overexpressed E7 to specifically decrease pRB level.…”

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

“…When expressed in transfected cells from a plasmid, NS forms inclusions similar to VFs that are termed viral factory-like (VFL) structures (9). Moreover, NS formation of VFLs results in specific VFL localization of each of the five viral structural proteins that make up the core particle ( 1, 2, 3, 2, and 2), the nonstructural NS protein that is involved in virus translation and replication, as well as the intact core particle itself (6,(9)(10)(11). The mechanism of VFL formation by NS is not fully understood; however, several regions of the protein, including the carboxyl (C)-terminal 7 amino acids (aa) and a putative metal chelating structure formed by amino acids His570 and Cys572, have been shown to be necessary for VFL formation in transfected cells and replication in infected cells (12)(13)(14).…”