title: The cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology.
abstract: The coronavirus nucleocapsid (N) protein plays a multifunctional role in the virus life cycle, from regulation of replication and transcription and genome packaging to modulation of host cell processes. These functions are likely to be facilitated by interactions with host cell proteins. The potential interactome of the infectious bronchitis virus (IBV) N protein was mapped using stable isotope labeling with amino acids in cell culture (SILAC) coupled to a green fluorescent protein-nanotrap pulldown methodology and liquid chromatography-tandem mass spectrometry. The addition of the SILAC label allowed discrimination of proteins that were likely to specifically bind to the N protein over background binding. Overall, 142 cellular proteins were selected as potentially binding to the N protein, many as part of larger possible complexes. These included ribosomal proteins, nucleolar proteins, translation initiation factors, helicases, and hnRNPs. The association of selected cellular proteins with IBV N protein was confirmed by immunoblotting, cosedimentation, and confocal microscopy. Further, the localization of selected proteins in IBV-infected cells as well as their activity during virus infection was assessed by small interfering RNA-mediated depletion, demonstrating the functional importance of cellular proteins in the biology of IBV. This interactome not only confirms previous observations made with other coronavirus and IBV N proteins with both overexpressed proteins and infectious virus but also provides novel data that can be exploited to understand the interaction between the virus and the host cell.
authors: Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors: Edward
authors: E
authors: Emmott
authors: Diane
authors: D
authors: Munday
authors: Erica
authors: E
authors: Bickerton
authors: Paul
authors: P
authors: Britton
authors: Mark A
authors: MA
authors: Rodgers
authors: Adrian
authors: A
authors: Whitehouse
authors: En-Min
authors: EM
authors: Zhou
authors: Julian A
authors: JA
authors: Hiscox
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D000086462:Coronavirus Nucleocapsid Proteins; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000502:physiology*; D006801:Humans; D001351:Infectious bronchitis virus / Q000235:genetics / Q000502:physiology*; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014709:Vero Cells
chemical_list: D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D020543:Proteome; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
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