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.

Fig 1

(A) Strategy for analyzing cellular interacting partners of IBV N protein via immunoprecipitation of an EGFP-N protein expressed in cells labeled by SILAC. Immunoprecipitations (IPs) were carried out using GFP-trap beads to minimize nonspecific binding. Labeling by SILAC was employed, as it allowed less stringent wash conditions to be employed, as contaminating proteins should be present in roughly equal amounts in both samples. Proteins showing an increased abundance with EGFP-N as the bait versus EGFP as the control bait are more likely to represent true interactions. Various stages in this process are highlighted. (B) Silver-stained SDS-polyacrylamide gel showing EGFP and EGFP-N pulldowns at various salt concentrations (indicated). (C). Immunoblot analysis of EGFP and EGFP-N pulldowns at various salt concentrations (indicated) to test the effects of buffer conditions on protein binding. Numbers to the left of the gels are molecular masses (in kilodaltons).

Fig 2

Bioinformatic analysis of the EGFP-N protein interactome using ingenuity pathway analysis showing the data organized into functional groupings in the cell. Numbers in parentheses indicate the number of proteins identified in each grouping.

Fig 3

STRING (version 9.0) analysis of the cellular proteins identified by the SILAC pulldown approach to be potentially interacting with IBV N protein. Proteins group into two large units: those associated with translation (right) and those involved in RNA modification/processing (left).

Fig 4

Immunoblotting confirmation of the interactions of identified cellular proteins with EGFP-N protein. (A) Agarose electrophoresis analysis of a small aliquot of the input lysate (to be used in the pulldown assay) in the absence (−) and presence (+) of RNase. Lane M, a 100-bp DNA ladder. (B) The pulldown experiment was repeated in the presence and absence of RNase and immunoblot analysis of selected cellular proteins.

Fig 5

Cosedimentation of EGFP-N protein with the small ribosomal subunit. Cosedimentation was performed on a 5 to 20% sucrose gradient in the presence of EDTA to induce separation of the large and small ribosomal subunits. The migration of these subunits was determined by extraction of RNA from the fractions and running of these on a 1% agarose gel containing ethidium bromide (EtBr), allowing visualization of the 18S and 28S rRNAs. Migration of the EGFP control or EGFP-N protein was determined by immunoblotting using an anti-GFP antibody.

Fig 6

Indirect immunofluorescent confocal microscopy showing colocalization of EGFP-N protein with the stress granule marker in G3BP in arsenite-stressed 293T cells. Cells were mock treated (−) or treated (+) with 0.5 mM sodium arsenite for 1 h prior to fixation. Nuclei are colored blue (DAPI [4′,6-diamidino-2-phenylindole]), EGFP is in green, G3BP is in red, and a merge image is presented. Colocalization is suggested by a yellow signal. Bars, 10 mm.

Fig 7

Indirect immunofluorescent confocal microscopy analysis of the localization of selected cellular proteins that potentially interact with N protein in IBV-infected cells. Cellular proteins are shown in red, IBV-infected cells are shown in green, and a merged image is presented. Colocalization is suggested by a yellow signal. Bars, 10 mm.

Fig 7

Indirect immunofluorescent confocal microscopy analysis of the localization of selected cellular proteins that potentially interact with N protein in IBV-infected cells. Cellular proteins are shown in red, IBV-infected cells are shown in green, and a merged image is presented. Colocalization is suggested by a yellow signal. Bars, 10 mm.

Fig 8

Investigation of the proviral/antiviral activity of selected cellular proteins that potentially interact with N protein in IBV-infected Vero cells. (A) Representative immunoblots showing the abundance of the target protein and IBV N protein in mock-infected and infected Vero cells either treated with siRNAs targeting the specific mRNA of interest or treated with a nontarget siRNA control. (B) MTT cell viability assay of Vero cells treated with either the nontarget siRNA or the specific siRNA to the mRNAs encoding the selected protein of interest. The experiment was performed three times in triplicate for each condition. (C) qRT-PCR analysis of the abundance of the IBV genomic RNA in either mock-infected cells (mock) or cells treated with the nontarget siRNA or the siRNA specific to the mRNAs encoding the selected protein of interest. Replication efficiency is related to the level observed in untreated cells. si-non target, nontargeting siRNA; si-Nucleolin, siRNA targeting nucleolin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; si-PARP1, siRNA targeting PARP1.