A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication

Figures

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  Fig 1. Characterization of the APEX2-GARG-1060 proximity biotinylation system. A. Domain organization of GBF1 and the C-terminally truncated GBF1 constructs fused to EGFP (positive control) and APEX2. Both GBF1 truncated constructs contain the BFA-resistant Sec7 domain from ARNO. B. Polio replicon replication was assessed in the presence or absence of 2μg/ml of BFA in HeLa cells transfected with plasmids expressing the C-terminally truncated GBF1 fusions with APEX2 or EGFP, or an empty vector C. Polio replicon replication assay was performed in control HeLa cells, or HeLa cells stably expressing APEX2-GARG-1060 with or without 2μg/ml of BFA. D. HeLa cells stably expressing APEX2-GARG-1060 were infected (or mock-infected) with 10 PFU/cell of poliovirus, and the biotinylation reaction was performed at 4 h p.i. The cells were processed for visualization of biotinylated proteins with a fluorescent streptavidin conjugate and immunostaining for a poliovirus antigen 3A. E. HeLa cells stably expressing APEX2-GARG-1060 were infected (or mock-infected) with poliovirus and the biotinylation reaction was performed as in D. The cells were stained with a fluorescent streptavidin conjugate and…

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  Fig 2. Known cellular proteins recruited to replication organelles are biotinylated by FLAG-APEX2-GARG1060. A. HeLa cells stably expressing FLAG-APEX2-GARG-1060 were infected (PV), or mock-infected (M) with 10 PFU/cell of poliovirus, and biotinylation was performed at the indicated times post-infection. Biotinylated proteins were collected on streptavidin beads, resolved on SDS-PAGE and analyzed in a Western blot with HRP-conjugated streptavidin. B. Scheme of the biotinylation experiment to compare the biotinylated proteins (strep pull-down) with the proteins in the lysates before fractionation (input). The figure was made using Biorender templates. C. HeLa cells stably expressing APEX2-GARG-1060 were infected (PV), or mock-infected (M) with 10 PFU/cell of poliovirus, the biotinylation reactions were performed at 2, 4, and 6 h p.i., and the unfractionated cellular lysates (input) and the isolated biotinylated proteins (strep pull-down) were analyzed in a western blot with the indicated antibodies. Anti-FLAG antibodies detect the APEX2-GARG-1060 protein.

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  Fig 3. Biotinylation of viral proteins by APEX2-GARG-1060. A. Poliovirus genome and polyprotein processing scheme. The cleavage sites for the viral proteases 2A, 3C, and 3CD are indicated by green, red, and blue-filled triangles, respectively. The dashed empty green triangle indicates a 2A cleavage site in 3D believed to be dispensable for replication. The purple star indicates the autocatalytic cleavage site in VP0. B. HeLa cells stably expressing APEX2-GARG-1060 were infected (PV), or mock-infected (M) with 10 PFU/cell of poliovirus, and biotinylation reactions were performed at the indicated times post-infection. Unfractionated cellular lysates (input) and isolated biotinylated proteins were analyzed in a Western blot with indicated antibodies. The antibodies recognize the final and intermediate polyprotein cleavage products containing the corresponding antigen. Red stars on anti-3A and anti-3D panels indicate polyprotein fragments that do not match the known polyprotein cleavage products.

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  Fig 4. Characterization of the proteome biotinylated by FLAG-APEX2-GARG1060. A. In five independent experiments, HeLa cells stably expressing APEX2-GARG-1060 were infected (PV), or mock-infected (M) with 10 PFU/cell of poliovirus, and biotinylation was performed at 6 h p.i. Biotinylated proteins were purified on streptavidin beads and analyzed in a Western blot with HRP-streptavidin. Biotinylated proteins from these five experiments were pooled for proteomics analysis. Full proteomics data are available in S1 Data. B. Gene ontology (GO) enrichment analysis of the proteomics data using PANTHER classification system [69]. Buble graphs show the number of proteins associated with a particular GO term (bubble size), the log2 of enrichment over the expected non-specific associations of genes in the dataset with a particular GO term (x-axis), and the statistical significance of the observed enrichment (negative log10 of p-value, y-axis). Five of the most statistically significantly enriched GO terms for proteins from infected and mock-infected samples are shown. The full GO analysis is available in S2 Data. C. The distribution of the poliovirus-specific peptides identified by mass-spectrometry…

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  Fig 5. Effects of siRNA-mediated knockdown of expression of AldoA, EWSR1 and ILF3-90 on enterovirus replication. A, B. HeLa cells were transfected with siRNAs specific to AldoA, EWSR1 and 90KDa isoform of ILF3, or non-targeting control siRNA, and polio or Coxsakie B3 replicon replication assays were performed 72 h post siRNA transfection. The total replication signal was calculated as the area under the corresponding kinetics curves. Cell viability signal is proportional to the level of ATP in cells. Western blots show the efficacy of siRNA-mediated knockdown of the targeted proteins. C. HeLa cells were transfected with siRNAs specific to AldoA, EWSR1 and 90KDa isoform of ILF3, or non-targeting control siRNA. 72 h post siRNA transfection cells were infected with an MOI of 1 PFU/cell of poliovirus or Coxsackie virus B3, and the total virus yield was determined at 6 h p. i. Western blots show the efficacy of siRNA-mediated knockdown of the targeted proteins. D. HeLa cells were transfected with siRNAs specific to 90KDa isoform of ILF3 or EWSR1 or a non-targeting control siRNA. 72h post siRNA transfection, the cells were transfected with a replication-defective replicon RNA containing the…

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  Fig 6. AldoA localization in infected and mock-infected cells. A, B. Confocal images of HeLa cells infected (or mock-infected) with 10 PFU/cell of poliovirus, fixed at 4 h p.i. and stained with antibodies against AldoA and a viral antigen 2B (A), or AldoA and dsRNA (B). Scale bar is 10 μm.

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  Fig 7. Cytoplasmic translocation of EWSR1 and its association with stress granules upon poliovirus infection. A. Confocal images of HeLa cells infected (or mock-infected) with 10 PFU/cell of poliovirus, fixed at 2, 4 and 6 h p.i and stained with antibodies against EWSR1 and the viral replication antigen 3B. B. High magnification confocal images of HeLa cells infected (or mock-infected) and processed as in A at 4 h p.i. Note the association of cytoplasmic EWSR1 signal outside of stress granules with the 3B-positive structures. C. Confocal images of HeLa cells infected (or mock-infected) as in A, fixed at 4 h.i. and stained with antibodies against EWSR1 and the stress granule component G3BP1. Scale bar is 10 μm. D. Confocal images of HeLa cells stably expressing APEX2-GARG-1060 infected (or mock-infected) as in A. C, and processed at 4 h p.i. to visualize biotinylated proteins and stained with antibodies to the stress granule component G3BP1. Scale bar is 5 μm.

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  Fig 8. ILF3-90 associates with dsRNA in poliovirus-infected cells. A, B. Confocal images of HeLa cells infected (or mock-infected) with 10 PFU/cell of poliovirus, fixed at 4 h p.i. and stained with antibodies against ILF3-90 and the viral antigen 2B (A), or ILF3-90 and dsRNA (B). Scale bar is 10 μm. Note the concentration of ILF3-90 signal on the outer border of the replication organelles and its association with dsRNA inside the replication organelles.