Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling
- Institute of Virology and Immunology IVI, Switzerland
- University of Bern, Switzerland
Figures
Figure 1 with 3 supplements
Characterization of the recombinant MHV-BirAR118G-nsp2.
(a) Genome organization of recombinant MHV-BirAR118G-nsp2. The positive-sense RNA genome of MHV contains a 5’ cap and a 3’ poly(A) tail. ORF1a and ORF1b encode the viral replication and …
Figure 1—figure supplement 1
Western blot detection of BirAR118G-nsp2.
MHV-BirAR118G-nsp2, MHV-A59- or non-infected L929 fibroblasts were cultured in medium or in medium supplemented with 67 µM biotin for 15 hr. Lysates were separated by SDS-PAGE and western blots were …
Figure 1—figure supplement 2
Immunofluorescence analysis of MHV-BirAR118G-nsp2-mediated biotinylation.
MHV-BirAR118G-nsp2, MHV-A59- or non-infected L929 fibroblasts were cultured in medium supplemented with 67 µM biotin. Cells were fixed 9 and 15 hr post infection (h.p.i.) and processed for …
Figure 1—figure supplement 3
Immunofluorescence analysis of the MHV-BirAR118G-nsp2 RTC and BirAR118G-nsp2 –mediated biotinylataion.
MHV-BirAR118G-nsp2, MHV-A59- or non-infected L929 fibroblasts were cultured in medium supplemented with 67 µM biotin. Cells were fixed 12 hr post-infection (h.p.i.) and processed for …
Figure 2
Determination of the coronavirus RTC-proximal proteome.
(a) Schematic overview of the BirAR118G-mediated proximity biotinylation assay using MHV-BirAR118G-nsp2. (b) Western blot analysis of MHV-BirAR118G-nsp2-infected L929 cells. L929 cells were infected …
Figure 3 with 1 supplement
Functional classification of RTC-proximal host factors.
(a) Gene Ontology enrichment analysis of RTC-proximal cellular factors. 32 terms were highly significant (p-value < 0.005) and were assigned to five broad functional categories: cell-cell adhesion, …
Figure 3—figure supplement 1
STRING protein interaction network analysis of the categories ‘cell organization’ (a) and ‘catabolic processes’ (b).
The nodes represent RTC-proximal host proteins and the edges represent the interactions, either direct (physical) or indirect (functional), between two proteins in the network.
Figure 4 with 1 supplement
Identification of proviral factors within the coronavirus RTC microenvironment.
(a) Impact of siRNA-silencing of RTC-proximal cellular proteins on viral replication. L929 fibroblasts were reverse-transfected with siRNAs (10 nM) for 48 hr before being infected with MHV-Gluc (MOI …
Figure 4—figure supplement 1
(a) siRNA controls contained in each 96-well plate during siRNA-silencing of the RTC-proximal library.
Controls included the established factors such as MHV entry receptor (Ceacam1a), Gbf1, Arf1. Arfgap2 was found to moderately affect MHV replication during pilot experiments and was included to cover …
Figure 5
Knockdown of eIF3e, eIF3f and eIF3i impact MHV replication.
(a) siRNA silencing and western blot assessment of eIF3e, eIF3f and eIF3i protein levels. 10 nM individual target-specific siRNA were transfected in L929 for 48 hr. Lysates were separated by …
Figure 6
Active translation near sites of MHV mRNA synthesis.
Visualization of active translation in MHV-infected L929 fibroblasts. Cells infected with MHV-A59 (MOI = 1) or non-infected cells were cultured for 6, 8, 10 and 12 hr and pulsed with cycloheximide, …
Figure 7 with 1 supplement
Active translation near sites of HCoV-229E and MERS-CoV mRNA synthesis.
Visualization of active translation during HCoV-229E and MERS-CoV infections. Huh7 cells were infected with HCoV-229E and MERS-CoV (MOI = 1) for 12 hr and 6 hr, respectively. Cells were pulsed with …
Figure 7—figure supplement 1
Visualization of active translation during HCoV-229E infections.
Huh7 cells were infected with HCoV-229E (MOI = 1) for 12, 15, 18, 24 hr. Cells were pulsed with cycloheximide, emetine and puromycin for 5 min to label translating ribosomes and subjected to a …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Escherichia coli) | BirAR118G | PMCID: 3308701 | ||
| Strain, strain background (mouse hepatitis virus) | MHV-A59 | PMID: 15709029 | ||
| Strain, strain background (mouse hepatitis virus) | MHV-Gluc | PMID: 24874215 | ||
| Strain, strain background (mouse hepatitis virus) | MHV-BirAR118G-nsp2 | This study | ||
| Strain, strain background (human coronavirus) | HCoV-229E | PMID: 19057873 | ||
| Strain, strain background (middle east respiratory syndrome coronavirus) | MERS-CoV | PMID: 23170002; 23078800 | ||
| Cell line (Mus musculus) | L929 | Sigma | 85011425 | |
| Cell line (Mus musculus) | 17Cl1 | Gift from S.G. Sawicki | PMC422565 | |
| Cell line (Homo sapiens) | Huh7 | Gift from V. Lohmann | CVCL_0336 | |
| Cell line (african green monkey) | Vero B4 | Gift from M. Müller | CVCL_1912 | |
| Antibody | anti-dsRNA J2 (mouse monoclonal IgG2a, kappa chain) | English and Scientific Consulting | Product No: 10010500 | 1:200 |
| Antibody | anti-myc (mouse monoclonal) | Cell signalling | 2276 | 1:8000 (IF); 1:1000 (WB) |
| Antibody | Anti-Nogo A + B (rabbit polyclonal) | Abcam | Product No : ab47085 | 1:200 |
| Antibody | Anti-EIF3E (rabbit polyclonal) | Sigma | HPA023973 | 1:100 (IF); 1:300 (WB) |
| Antibody | Anti-EIF3F (rabbit polyclonal) | Abcam | ab176853 | 1:3000 (WB) |
| Antibody | Anti-EIF3I (rabbit polyclonal) | Sigma | HPA029939 | 1:500 (WB) |
| Antibody | Anti-Puromycin(mouse monoclonal IgG2a,κ) | Merk Millipore | MABE343 | 1:10000 |
| Antibody | Anti-MHV nsp2/3 (rabbit polyclonal) | Gift from S. Baker | PMID: 9514967 | 1:200 |
| Antibody | Anti-MHV nsp8 (rabbit polyclonal) | Gift from S. Baker | PMID: 11907209 | 1:400 |
| Antibody | Anti-229E-nsp8 (rabbit polyclonal) | Gift from J Ziebuhr | PMID: 9847320 | 1:200 |
| Antibody | donkey anti-mouse 488 | Jackson ImmunoResearch | 715-545-150 | 1:400 |
| Antibody | donkey anti-rabbit 594 | Jackson ImmunoResearch | 711-585-152 | 1:400 |
| Antibody | donkey anti-rabbit 647 | Jackson ImmunoResearch | 711-605-152 | 1:400 |
| Antibody | donkey anti-rabbit HRP | Jackson ImmunoResearch | 711-035-152 | 1:10000 |
| Antibody | donkey anti-mouse HRP | Jackson ImmunoResearch | 715-035-151 | 1:5000 |
| Antibody | anti-actin HRP (mouse monoclonal) | Sigma | A3854 | 1:25000-1:50000 |
| Sequence-based reagent | On-Target Plus CherryPick siRNA Library | Horizon Discovery Ltd. | ||
| Commercial assay or kit | Pierce Gaussia Luciferase Glow Assay Kit | ThermoFisher Scientific | 16160 | |
| Commercial assay or kit | CytoTox 96 Non-Radioactive Cytotoxicity Assay | Promega | G1780 | |
| Commercial assay or kit | Viromer Green | Lipocalyx | VG-01LB-00 | |
| Commercial assay or kit | Dynabeads MyOne Streptavidin C1 | ThermoFisher Scientific | 65001 | |
| Chemical compound, drug | Puromycin | Sigma | P9620 | |
| Chemical compound, drug | Cycloheximide | Sigma | C7698 | |
| Chemical compound, drug | Emetin | Sigma | E2375 | |
| Chemical compound, drug | Biotin | Sigma | B4501 |
Additional files
-
Supplementary file 1
Mass spectrometry data.
- https://doi.org/10.7554/eLife.42037.016
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Supplementary file 2
Gene Ontology enrichment analysis and STRING functional protein association association network analysis.
- https://doi.org/10.7554/eLife.42037.017
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Supplementary file 3
siRNA-mediated knockdown screen.
- https://doi.org/10.7554/eLife.42037.018
-
Supplementary file 4
Primer and probes used in RT-qPCR.
- https://doi.org/10.7554/eLife.42037.019
-
Transparent reporting form
- https://doi.org/10.7554/eLife.42037.020
