The Cellular Interactome of the Coronavirus Infectious Bronchitis Virus Nucleocapsid Protein and Functional Implications for Virus Biology

Determination of the interactome of non-structural protein12 from highly pathogenic porcine reproductive and respiratory syndrome virus with host cellular proteins using high throughput proteomics and identification of HSP70 as a cellular factor for virus replication

“…ACCEPTED MANUSCRIPT 16 Similarly, MKT-077 treatment also caused the reduction of NSP12-GFP, and the protein level was recoverable with the presence of proteasome inhibitor (Figure. 7).…”

“…The interactome of PRRSV NSP12 was determined by high-affinity GFP pull-down coupled with label free mass spectrometry-based approach, which we have used previously to study virus/host protein interactions [16][17][18][19]. Our data identified112 cellular proteins probably interacted with NSP12, and these proteins were mainly nucleic acid binding proteins or chaperones and were grouped into several distinct functional clusters.…”

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin

“…In recent years, a high-throughput, quantitative proteomic SILAC-based technique has been applied to study interactomes between viral proteins and host cellular proteins [41]. Most of these studies investigated the interactions between the viral and cellular proteins by using the overexpression of a target viral protein fused with a tag, such as green fluorescent protein in combination with a SILAC-based LC-MS/MS approach, coupled with immunoprecipitation and tag-trap beads [32,42]. This approach allows us to distinguish between cellular proteins that specifically bind to the viral protein and background binding proteins.…”

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

“…This has advantages over the yeast two-hybrid approach in that cell localization and post-translational modifications are not perturbed, as well as advantages over traditional TAP-tagging in that it is a quantitative rather than qualitative approach allowing the user to readily distinguish non-specifically interacting proteins and contaminants, from host factors that bind specifically. Further, as a sample is typically analyzed whole, rather than as individual protein bands, proteins of interest are not masked by similarly migrating proteins on a gel, nor do they typically need to be present at sufficient levels to be visible after staining, leading to increased numbers of confidently identified proteins 4 .…”

“…The results of this analysis are then processed to identify high confidence protein:protein interactions (Figure 1). SILAC immunoprecipitation enables the identification of not only direct interactions but also low affinity or indirect interactions with protein complexes 4 . Using this system, eIF4AI and II immunoprecipitations allowed reproducible and confident identification of the primary binding partner eIF4G (isoforms I/II and III)…”