Fig 1.
Characterization of recombinant M2-2 expression in human cell lines.
(A) Detection of FLAG and EGFP-M2-2 expression in HEK293T cells at 24 hpt. FLAG-EGFP was used as a control for protein detection. Observed molecular weights are shown on the left. (B) Immunofluorescence of FLAG-M2-2 and EGFP-M2-2 expressed for 24h in HEp-2 cells. Panels show distinct distribution patterns observed for both proteins, as indicated on the left. (C) Confocal immunofluorescence showing co-expression of FLAG-M2-2 and EGFP-M2-2 in the same cell (second row) or expressed alone (first row). All images are representative of at least three independent experiments. Scale bars 10 μm.
Fig 2.
Expression of tagged M2-2 proteins in RSV infected human cells.
(A) Expression of FLAG-M2-2 or EGFP-M2-2 (B) in mock or RSV infected HEp-2 cells. Images show the recruitment of M2-2 to the vicinity of viral IBs, stained with anti-N, as evidenced on zoomed areas. FLAG and EGFP-M2-2 localization to the nucleus and cytoplasmic granules is also observed. Raw confocal images were submitted to deconvolution as described in the methods section. All images are representative of at least three independent experiments. Scale bars 10 μm.
Fig 3.
Proteomic analysis of M2-2 interactors shows enrichment of ribosome components, chaperones and splicing factors.
(A) FLAG-M2-2 or empty vector were expressed in HEK293T cells for 48h and submitted to immunoprecipitation protocol. Western blot shows detection of FLAG-M2-2 in lysate (L) and eluted proteins (IP). Images are representative of three independent experiments sent for mass spectrometry analysis. (B) STRING network of the 72 proteins identified by mass spectrometry as potential interactors of M2-2. Lines connecting nodes indicate high-confidence interactions, as set during analysis. Protein names are indicated on nodes. Three main clusters are highlighted on the network, showing proteins related to translation, protein folding and mRNA splicing. (C) Graph showing enriched GO terms for Biological Process. Enrichment analysis for the identified proteins was performed on BinGO, as described in the methods section.
Fig 4.
M2-2 does not colocalize with G3BP1 and stress granules.
Expression of FLAG-M2-2 (A) and EGFP-M2-2 (B) in HEp-2 cells stained with anti-G3BP1. Cells were treated with 200 nM ISRIB (for 20h) or 0.5 mM arsenite for 30 min before fixation. Non-treated (NT) and ISRIB cells show diffuse G3BP1 distribution, while SGs can be observed under arsenite treatment. Distinction between M2-2 granules and SGs can be seen in zoomed areas. All images were taken with a Zeiss LSM-780-NLO microscope and are representative of three independent experiments. Scale bars 10 μm.
Fig 5.
M2-2 expression inhibits SGs assembly and eiF2α phosphorylation.
(A) Immunofluorescence of FLAG-EGFP, FLAG-M2-2 and EGFP-M2-2 in HEp-2 cells treated with arsenite (0.5 mM for 30 min). Inhibition of SGs assembly, stained by G3BP1, can be seen in cells expressing M2-2. (B) The graph presents the average proportion of cells shown in (A) positive for stress granules, showing reduction in SG assembly during the expression of M2-2. Roughly 30 cells were counted in each independent experiment, and colored dots indicate individual values for each experiment (n = 3). Error bars show standard deviation. (C) FLAG-M2-2 and EGFP-M2-2 or controls (FLAG-EGFP and empty vector) were transfected in HEK293T cells, and after 24h cells were lysed and analyzed by western blot. Treatments with ISRIB (200 nM) or arsenite (0.5 mM) were performed as previously described. Phosphorylation of eiF2α was quantified and submitted to one-way anova followed by Tukey’s multiple comparisons test (D). Graph presents mean values, with colored dots indicating values from independent replicates (n = 3). Error bars show standard deviation and p-values are shown in the graph.
Fig 6.
M2-2 expression downregulates translation.
(A) SUnSET assay performed in HEK293T cells expressing the control FLAG-EGFP, or FLAG and EGFP-M2-2 proteins. After 24h, cells were incubated with puromycin (10 μg/mL) for 10 min, rinsed and lysed for western blot analysis. Puromycin together with cycloheximide (100 μg/mL) or non-treated cells were used as controls. Mean translation rates, indicated by puromycin staining, are quantified in (B), and all images are representative of three independent experiments (n = 3). (C) Reporter luciferase assay evaluating the inhibitory effect of M2-2 in the translation of Renilla (5’ cap) and Firefly (IRES) luciferases, as shown above. Graphs show means of the relative luminescence units (RLU) obtained in three independent experiments performed in triplicate (n = 3). Colored dots shown in (B) and (C) indicate individual means obtained in each individual experiment. Statistical analysis was performed by one-way anova followed by Dunnett’s multiple comparisons test. Error bars indicate standard deviation and p-values are indicated on the graph.
Fig 7.
Proteasome and HSP70 inhibition promote enhanced detection of M2-2 by western blot.
(A) Western blot detection of FLAG-EGFP, FLAG-M2-2 and EGFP-M2-2 expressed for 24h in HEK293T cells non-treated (NT), or under ISRIB (200 nM) and MG132 (5 μM) treatments. Images are representative of three independent experiments. Graphs in (B) present the mean values quantified in (A). Statistical analysis was performed by one-way anova followed by Dunnett’s multiple comparisons test and p-values are indicated on the graph. (C) Expression of FLAG and EGFP-M2-2 in HEp-2 cells, as described for HEK293T cells. Normalized expression of FLAG and EGFP-M2-2 is shown below. (D) Expression of FLAG and EGFP-M2-2 under VER treatment (NT, 2.5 or 5 μM). Western blots are quantified in (E) and are representative of three independent experiments. In the graphs, colored dots indicate values from independent replicates (n = 3), and error bars show standard deviation.
Fig 8.
MG132 treatment induces the recruitment of FLAG-M2-2 into cytoplasmic granules.
Cellular distribution of FLAG-M2-2 and EGFP-M2-2 under different treatments (ISRIB 200 nM for 20h, MG132 5 μM for 2h) in HEp-2 (A) or A549 cells (B), showing increase in the number of granules stained by FLAG-M2-2 under MG132 treatment. Graphs in (C) and (D) present the quantification in HEp-2 cells of the average number of granules for FLAG and EGFP-M2-2 during each treatment. Small dots show the individual number of counted granules over 30 cells for each condition, while bigger dots present mean values. Different colors show values obtained across each independent experiment (n = 3). Statistical differences were evaluated by one-way anova following Tukey’s multiple comparisons test. Error bars indicate standard deviation, and p-values are indicated by * p < 0.05 and *** p < 0.001, ns = not significant.
Fig 9.
M2-2 interacts with defective proteins targeted for proteasome degradation.
FLAG-M2-2 (A) and EGFP-M2-2 (B) were expressed in HEp-2 cells for 24h, and then cells were incubated with puromycin (10 μg/mL) and MG132 (5 μM) for 2h. After treatment, cells were rinsed, fixed, and stained with anti-puromycin for tracking of DRiPs, showing colocalization with M2-2. Inhibition of translation can be observed in cells presenting low puromycin incorporation (white arrows in (A) and (B)–third row). (C) As a control, FLAG-M2-1 was expressed and submitted to the same treatments. White arrows in the zoomed area show absence of colocalization between puromycin stained granules and FLAG-M2-1. All images were taken with a Zeiss LSM-780-NLO microscope and are representative of three independent experiments. Scales bars are 10 μm for full size panels, and 2 μm for zoomed panels.
Fig 10.
M2-2 shows proximity with the ribosomal machinery.
(A) HEK293T cells expressing FLAG-EGFP, FLAG-M2-2 (B) or EGFP-M2-2 (C) for 24h were submitted to cell fractionation through sucrose gradient. Staining with the proteins GAPDH and S6 were used to determine soluble and ribosomal fractions. Images show that FLAG and EGFP-M2-2 can be found in light ribosomal fractions, as highlighted in the blots (fractions 4–6). Otherwise, FLAG-EGFP were detected only in soluble fractions (1–3). (D) FLAG-M2-2 and EGFP-M2-2 were expressed in HEp-2 or Vero E6 cells (E) and analyzed by confocal immunofluorescence against the ER marker calreticulin, showing colocalization between the proteins and this structure. Images are representative of three independent experiments. Zoomed areas in (E) show the proximity between M2-2 and calreticulin in higher detail. Scale bars are 10 μm for full size panels and 1 μm for zoomed areas.