Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation
- University of Cambridge, United Kingdom
- Washington University School of Medicine, United States
- Harvard Medical School, United States
- South China Normal University, China
- Northeastern University, United States
- Karolinska Institute, Sweden
- University of Surrey, United Kingdom
- Yale School of Medicine, United States
Figures
Figure 1 with 1 supplement
The norovirus VPg proteins interacts with ribosome associated translation initiation factors.
(A) Amino acid sequence alignment of the GV murine norovirus VPg sequences with VPg from representative human noroviruses from GI Norwalk virus (NV), GII, and GIV. The position of the site of RNA …
Figure 1—figure supplement 1
Host factors binding to the norovirus VPg.
Quantitative proteomics was used as described in the text to identify host factors that were affinity purified following transfection of GFP-tagged derivative of either the NV or MNV VPg proteins. …
Figure 2 with 2 supplements
Proteomic characterisation of the norovirus replication complex using infectious epitope tagged MNV.
(A) Schematic representation of NS1/2-FLAG and NS4-FLAG viruses contain insertions of nucleotide sequences encoding the FLAG peptide DYKDDDDK (in yellow) in their coding sequences. The NS1/2-FLAG …
Figure 2—figure supplement 1
Additional analyses of NS1/2 and NS4-associated proteins.
MNV proteins highlighted in light blue, and G3BP1 in gold. (A) Gene ontology using PANTHER overrepresentation analysis of proteins copurifying with NS1/2 or NS4 (Log2 SILAC ratio >2 for either …
Figure 2—figure supplement 2
Further analysis of proteins enriched through MNV VPg proteomics and FLAG-tagged virus replication complex proteome data (Log2 SILAC ratio >1).
(A) Venn diagram illustrating the degree of overlap between the proteins identified in the three proteomics approaches. The greatest overlap between VPg and the replication complex proteome data was …
Figure 3
CRISPR screen identifies host genes positively and negatively selected upon MNV infection.
(A) Schematic overview of the infection CRISPR screen workflow. BV2 cells expressing Cas9 were transduced with a CRISPR library then subsequently infected with either MNV CR6 or CW3 for 24 hr. Cells …
Figure 4
CRISPR knockout of G3BP1 renders cells non permissive for MNV replication.
(A) Western blot analysis of three independent ΔG3BP1 clones for GAPDH, G3BP1 and G3BP2. (B) High multiplicity, single cycle growth curve analysis of the impact of G3BP1 ablation on MNV replication. …
Figure 5
G3BP1 is required for human Norwalk virus replication in cell culture.
(A) Colony formation ability of human norovirus VPg-linked RNA isolated from BHK-NV replicon containing cells is dependent on the presence of VPg. NV VPg-linked RNA isolated from BHK-NV cells was …
Figure 6
MNV replication in BV2 cells requires the RNA binding activity of G3BP1.
(A) Schematic illustration of the G3BP1 truncations used to identify the domains involved in the norovirus life cycle. The positions of the various domains including the RRM and RGG domains deleted …
Figure 7
Loss of G3BP1 results in a defect following transfection of viral VPg-linked RNA into ΔG3BP1 cells.
(A) The indicated cell lines were transfected with MNV viral RNA and harvested at 9 hr post transfection for TCID50 to assess the virus yield. In some instances, the nucleoside analogue 2CMC was …
Figure 8
The Lack of G3BP1 results in a failure to produce viral negative sense RNA.
The experimental design is illustrated in (A). Wild type or ΔG3BP1 (1B2) cells were infected prior to the addition of the nucleoside analogue 2CMC to prevent viral RNA synthesis. Samples were …
Figure 9 with 1 supplement
G3BP1 is required for the association of VPg and norovirus VPg-linked RNA with ribosomal subunits.
(A) GFP-Trap immunoprecipitation of complexes isolated on with GFP alone or GFP tagged wild type MNV-VPg demonstrating the pull down of eIF4G1, G3BP1 and 40S subunits (RpS6). BV2 cells were …
Figure 9—figure supplement 1
G3BP1 is required for the association of VPg with ribosomal subunits.
GFP-Trap pull down was performed as described in Figure 9 following transfection of GFP tagged derivatives of either a WT or a F123 MNV VPg proteins into WT BV2 cells, ΔG3BP1 BV2 cells or ΔG3BP1 BV2 …
Figure 10
G3BP1 is required for polysome association of viral RNA association.
(A) Polysome profiles of the ribosome containing fractions from mock or MNV infected wild type (WT) or ΔG3BP1 (1B2) BV2 cells at 4 and 9 hr post infection (moi 3 TCID50/cell). (B) Relative viral RNA …
Figure 11 with 1 supplement
G3BP1 is required for efficient norovirus VPg-dependent translation.
(A) Illustration of the bicistronic construct used to assess the in vitro translation efficiency of the extracts prepared from WT BV2 cells or cells lacking G3BP1. The location of the 5’ cap and the …
Figure 11—figure supplement 1
Lack of G3BP-1 results in reduced Norovirus VPg-dependent translation efficiency in vitro.
(A) Characterisation of viral VPg-linked RNA. The sensitivity of purified MNV VPg-linked RNA to various nucleases was compared to in vitro transcribed capped MNV gRNA (cap-gRNA) and the MNV1 full …
Additional files
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Supplementary file 1
Raw data associated with Figure 1.
(A) Log2 SILAC ratio of proteins identified in Norwalk virus (NV) VPg GFP Trap pull downs. The experimental details are provided in the Materials and methods. SILAC ratios were computed by comparing the ratio of the peptides for each protein in the GFP control pull down to that obtained with the NV-GFP fusion protein. As described in the text each experiment was performed in three independent conditions varying the label in each biological sample. M/L – medium vs light sample, H/L – heavy vs light labelled sample and M/L – medium vs light sample. (B) As described in panel A but as obtained using the MNV VPg-GFP fusion protein as the bait.
- https://doi.org/10.7554/eLife.46681.019
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Supplementary file 2
Raw data associated with Figure 2.
(A) Log2 SILAC ratio of proteins identified anti-FLAG immunoprecipitations from cells infected with the NS1/2 tagged MNV. The experimental details are provided in the Materials and methods. SILAC ratios were computed by comparing the ratio of the peptides for each protein in the anti-Flag immunoprecipitations performed on cells infected with wild type MNV. As described in the text each experiment was performed in three independent conditions varying the label in each biological sample. M/L – medium vs light sample, H/L – heavy vs light labelled sample and M/L – medium vs light sample. The Log2 transformed values and the average values are shown. (B) As described in panel A but as obtained using the NS4-FLAG tagged MNV. C) Combined dataset obtained using NS1/2 and NS4 tagged viruses showing the average Log2 SILAC ratios.
- https://doi.org/10.7554/eLife.46681.020
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Supplementary file 3
Gene ontology and cross comparison analysis of the data obtained in Figure 2.
(A) Gene ontology of the host proteins found to be enriched by both NS1/2 and NS4. (B) PANTHER overexpression analysis of host proteins found to be enriched by both NS1/2 and NS4. (C) Raw data used for PANTHER analysis shown in panel B. (D) Colour code for data shown in panel C. (E) List of host proteins identified in previous studies as having (potential) roles in the norovirus life cycle along with their degree of overlap with the host proteins identified using NS1/2 and NS4. (F) Gene ontology analysis of host proteins identified as binding to the MNV NS1/2 protein and enriched using GFP-tagged MNV VPg.
- https://doi.org/10.7554/eLife.46681.021
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Supplementary file 4
Raw data and further analysis of the data obtained in Figure 3.
(A) List of genes ranked with a positive STARS obtained with the Brie CRISPR screen against MNV CW3. (B) List of genes ranked with a negative STARS obtained with the Brie CRISPR screen against MNV CW3. (C) As in panel A except using the MNV strain CR6. (D) As in panel B except using the MNV strain CR6. (E) Combined STARS ranking for the genes in both MNV CW3 and CR6 data sets. (F) Comparison of genes with positive and negative STARS values in the data sets obtained using MNV CW3 and CR6. (G) Comparison of data from panel A and C with the previous MNV CRISPR screens. (H) Gene ontology overexpression analysis of genes identified in this study as having positive STARS values for both MNV CW3 and CR6.
- https://doi.org/10.7554/eLife.46681.022
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Supplementary file 5
Comparison of data obtained from three screens to identify host factors involved in the norovirus life cycle.
(A) Comparison of the data obtained using MNV VPg-GFP trap with the MNV NS1/2 and NS4-FLAG tagged purifications. (B) Comparison of the data obtained using the CRISPR screen and the MNV NS1/2 and NS4-FLAG tagged purifications. (C) Comparison of the data obtained from all three screens.
- https://doi.org/10.7554/eLife.46681.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.46681.024
