Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation

Fig 1. CrPV-1A expression inhibits stress granules in response to arsenite treatment. (A) Depiction of the CrPV genome with the structure of CrPV-1A protein (PDB 6C3R) (below) highlighting the domains selected for mutagenesis. (B) Schematic of CrPV-1A-2A-GFP RNA containing the CrPV 5’ and 3’UTRs. (C) Confocal immunofluorescence images of S2 cells transfected with control 5’cap-GFP-poly (A)+, wild type or R146A mutant CrPV-1A-2A-GFP RNAs (16 hours) followed by one-hour treatment in the presence or absence of 500 μM sodium arsenite. The arrows show transfected cells. Shown are representative transfected cells detecting GFP fluorescence (green), Rin antibody staining (red), Hoechst dye staining for nucleus (blue) and merged images. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (D) Box plot showing the number Rin foci per cell. At least 50 cells were counted for each condition from three independent experiments. Data are mean ± SD. P > 0.05 (ns) p < 0.0001(****) by a one-way ANOVA (nonparametric) with a Bonferroni’s post hoc-test.

Fig 2. CrPV-1A mediated stress granule inhibition requires the BC Box domain and is independent of the Ago-2 binding domain. (A) Images of transiently transfected S2 cells with the indicated in vitro transcribed RNAs (16 hours), followed by one-hour sodium arsenite treatment (500 μM). Shown are representative transfected cells detecting GFP fluorescence (green), Rin antibody staining (red) and Hoechst dye staining for nucleus (blue) and merged images. The arrows show transfected cells. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (B) Box plot of the number of Rin foci per cell. At least 50 cells were counted for each condition from three independent experiments. Data are mean ± SD. p > 0.05 (ns), p < 0.021 (*),p < 0.0001(****) by a one-way ANOVA (nonparametric) with a Bonferroni’s post hoc-test.

Fig 3. R146 of CrPV-1A promotes full 2A peptide activity: (A) Immunoblots of lysates from S2 cells transfected with the indicated reporter RNAs (16 hours post transfection). A light exposure (top) and longer exposure (bottom) of an anti-GFP immunoblot and CrPV-1A immunoblot is shown. (B) Autoradiography of [35S] Met/Cys labelled proteins from in vitro sf-21 translation reactions incubated with the indicated RNAs. (C) Percent quantification of [35S] Met/Cys labelled CrPV-1A proteins. Data are mean ± SD from three independent experiments. p > 0.05 (ns), p < 0.002(**), p < 0.0001(****) by a one-way ANOVA (nonparametric) with a Bonferroni’s post hoc-test. (D) Immunoblots of lysates from S2 cells infected with (M) mock, CrPV or CrPV (R146A) (MOI 10) at indicated time points.

Fig 4. CrPV-1A localizes to the nucleus and induces poly(A)+ RNA accumulation in the nucleus. (A) Confocal immunofluorescence images of S2 cells transfected with in vitro transcribed RNA encoding CrPV-1A, CrPV-1A(R146A), or CrPV-1A(F114A) for 16 hours. GFP fluorescence (green), CrPV-1A antibody staining (red), fluorescence in situ hybridization using Cy5-oligo(dT) probes (cyan) and Hoechst dye (blue). The arrows show transfected cells. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (B) Box plot of the fraction of nuclear to total Cy5-oligo(dT) fluorescence intensity in each cell. At least 50 cells were counted for each condition from two independent experiments. Data are mean ± SD. p > 0.05 (ns), p < 0.021 (*), p < 0.002(**), p < 0.0001(****) by a one-way ANOVA (nonparametric) with a Bonferroni’s post hoc-test.

Fig 5. CrPV-1A localizes to the nucleus during virus infection. Z-stack confocal images of S2 cells infected with (A) wild-type CrPV or (B) CrPV(R146A) virus. From left to right are Z-stack images through the cells. Cells were fixed and stained with Lamin (green), CrPV-1A (red) and Hoechst (blue). Shown are representative images from three independent experiments.

Fig 6. Transcriptional profiling of CrPV and CrPV(R146A) infected S2 cells. (A) Principal component analysis of transcriptional signatures from cells infected with Mock, CrPV or CrPV(R146A). (B) Bar graph indicating the number of differentially expressed genes for each comparison identified by DESeq2 (C) Volcano plots showing changes in gene expression with fold change (FC) in expression intensity of DEGs, plotted against corresponding FDR for 2 hrs and (D) 4 hrs. (E) Network analysis diagram showing Gene ontology analysis filtered by molecular function for CrPV infected cells (F) Venn diagram showing comparison of dicistrovirus transcriptome datasets [100].

Fig 7. RNA export modulates CrPV infection. (A) Fluorescence in situ hybridization using Cy5-oligo(dT) (cyan) of S2 cells incubated with dsRNA targeting RNA export factor NXF1 or control GFP and Hoechst dye (blue). Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 3X zoom. (B) Box plot of the fraction of nuclear to total Cy5-oligo(dT) fluorescence intensity in each cell. At least 40 cells were counted for each condition from two independent experiments. Data are mean ± SD. p < 0.0001(****) by a student t-test. (C) Viral yield from wild-type and mutant (R146A) CrPV infected S2 cells for 8 hours (MOI 10) was accessed by fluorescence foci unit (FFU). Shown are averages from two independent experiments.

Fig 8. Nup358 promotes CrPV infection in an R146-dependent manner. (A) Immunoblots of S2 cells treated with dsRNA targeting GP210, mTor, Rae1, Nup88, Nup214, Nup358 or control FLuc, followed by mock infection or infection with wild-type or mutant CrPV virus for 8 hours (MOI 1). (B) Quantification of VP2 intensity normalized to tubulin from three independent experiments. The intensity values are normalized to the VP2/Tubulin intensity in FLuc control knockdown cells. (C) CrPV viral RNA levels by qRT-PCR analysis normalized to Rps9 mRNA levels. Data are mean ± SD relative to WT p > 0.05 (ns), p < 0.002(**), p < 0.0001(****) by a one-way ANOVA (nonparametric) with a Bonferroni’s post hoc test. (D) Immunoblot of S2 cell lysates treated with dsRNA targeting FLuc or Nup358. (E) FISH using Cy5-oligo(dT) or antibody staining of Rin (red) of S2 cells treated with control dsRNA or Nup358 dsRNA in the presence of DMSO or Pateamine A (Pat A). Hoechst staining is shown in blue. The arrows show Nup358-knockdown cells concomitant with nuclear poly(A)+ enrichment. Shown are representative images of at least two independent experiments. (F) Box plot of the fraction of nuclear to total Cy5-oligo(dT)…

Fig 9. Nup358 is degraded during CrPV infection. (A) Co-immunoprecipitation assay of S2 cells transiently transfected with FLAG-Nup358 for 24 hours, then transfected with either GFP, CrPV-1A, or CrPV-1A(R146A) RNA for 16 hours. Lysates were incubated with FLAG antibody as bait and precipitated using Protein A/G Magnetic beads. Shown are representative immunoblots from three independent experiments. (B) Immunoblots of S2 cell lysates infected with (M) mock, CrPV or CrPV(R146A) (MOI 1) in the presence or absence of DMSO or 50 μM MG132. Shown are representative immunoblots from three independent experiments.

Fig 10. Model of stress granule inhibition by CrPV-1A. During CrPV infection, CrPV-1A localizes to the nuclear membrane in an R146-dependent manner and recruits Cul2-Rbx1-EloBC complex to ubiquitinate Nup358 leading to its degradation. The degradation of Nup358 results in a block in mRNA export, resulting in the enrichment of poly (A)+ mRNAs in the nucleus, and together inhibits stress granule formation which, facilitates virus infection.