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. 2017 Nov 30;7(1):16652.
doi: 10.1038/s41598-017-16952-8.

Ribosomal stress and Tp53-mediated neuronal apoptosis in response to capsid protein of the Zika virus

Lukasz P Slomnicki  1 , Dong-Hoon Chung  2 , Austin Parker  1 , Taylor Hermann  1 , Nolan L Boyd  3 , Michal Hetman  4   5
Affiliations

Ribosomal stress and Tp53-mediated neuronal apoptosis in response to capsid protein of the Zika virus

Lukasz P Slomnicki et al. Sci Rep. .

Abstract

We report here that in rat and human neuroprogenitor cells as well as rat embryonic cortical neurons Zika virus (ZIKV) infection leads to ribosomal stress that is characterized by structural disruption of the nucleolus. The anti-nucleolar effects were most pronounced in postmitotic neurons. Moreover, in the latter system, nucleolar presence of ZIKV capsid protein (ZIKV-C) was associated with ribosomal stress and apoptosis. Deletion of 22 C-terminal residues of ZIKV-C prevented nucleolar localization, ribosomal stress and apoptosis. Consistent with a casual relationship between ZIKV-C-induced ribosomal stress and apoptosis, ZIKV-C-overexpressing neurons were protected by loss-of-function manipulations targeting the ribosomal stress effector Tp53 or knockdown of the ribosomal stress mediator RPL11. Finally, capsid protein of Dengue virus, but not West Nile virus, induced ribosomal stress and apoptosis. Thus, anti-nucleolar and pro-apoptotic effects of protein C are flavivirus-species specific. In the case of ZIKV, capsid protein-mediated ribosomal stress may contribute to neuronal death, neurodevelopmental disruption and microcephaly.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Nucleolar stress in ZIKV-infected neuroprogenitor cells. Freshly isolated rat embryonic NPCs (rNPCs) or monolayer cultures of human iPSC-derived NPCs (hNPCs) were infected with ZIKV strains MR766 or PRVABC59 at MOI 0.1. The rNPCs were grown as neurospheres for 3 days post infection (dpi), dispersed and cultured as a monolayer for 16 h to enable microscopic analysis at a single cell level; the hNPCs were maintained in a monolayer culture. After fixation, co-immunofluorescence staining was performed for the ZIKV infection marker (the flaviviral E protein, Flavi-E) and the nucleolar marker nucleophosmin-1 (NPM1/B23); nuclear DNA was counterstained with Hoechst-33258. Additional NPC data on ZIKV infection and cytotoxicity are presented in Supplemenary Figs S1 and S2. (a) Representative images depicting non-apoptotic ZIKV-infected rNPCs (i.e. lacking apoptotic chromatin condensation). Dotted lines mark nuclear contours of these cells; note reduced fluorescence intensity (FI) of NPM1. (b) At least MR766 infection increased fraction of non-apoptotic cells without NMP1-positive nucleoli. (c,d) Quantification of NPM1 signal confirmed ZIKV-induced reduction of fluorescence intensity (FI) as well as nucleolar territory. (e) Nucleolar number was unaffected by ZIKV. (f) Nucleolar stress in ZIKV-infected hNPCs as revealed by reduced nucleolar FI of NPM1 signal at dpi 1 (representative images are shown in Supplementary Fig. S2). (g) At dpi 4, there was also a significant reduction in NPM1-defined nucleolar territory of PRV-infected cells. (h) Nucleolar number was unaffected. At dpi 4, cells that survived MR766 infection showed similar anti-nucleolar effects as those infected with PRV (Fig. S3). Immunofluorescence staining for an additional marker of the nucleolar GC, PES1 confirmed negative effects of ZIKV infection on hNPC nucleoli (Supplementary Fig. S4). Data represent two independent experiments including 2 sister cultures/experiment in (b), and, at least 58 (c–e) or 27 (f–h) randomly selected individual cells with no signs of chromatin condensation that were analyzed for each condition; error bars are SEM. Data were analyzed by u-test (b) or one-way ANOVA and Tukey’s post-hoc tests (c-h), NS, p > 0.05; *p < 0.05; ***p < 0.001.
Figure 2
Figure 2
Pro-apoptotic and anti-nucleolar effects of ZIKV in rat embryonic cortical neurons. At day in vitro (DIV) 1, neurons were infected with ZIKV strains MR766 or PRVABC59 (PRV) at MOI 0.1. (a) Representative images of infected neurons that were identified by Flavi-E immunofluorescence; apoptotic condensation of nuclear chromatin was visualized by counterstaining DNA with Hoechst-33258. Arrows identify infected neurons with healthy nuclei; arrowheads indicate infected neurons with signs of apoptosis (condensation and/or fragmentation of chromatin). (b) Increased apoptosis of ZIKV-infected neurons. (c) Representative images depicting non-apoptotic, ZIKV-infected neurons (arrows) that were co-immunostained for the nucleolar marker NPM1. Note reduced fluorescence intensity (FI) of the nucleolar NPM1 signal, apparent shrinkage of NPM1-positive nucleolar territory and increased nucleoplasmic signal of NPM1 indicative of its release from nucleoli. (d) ZIKV MR766 infection increased fraction of non-apoptotic cells without NMP1-positive nucleoli. (e,f) Quantification of nucleolar NPM1 signal confirmed ZIKV-induced reduction of its intensity as well as territory. (g) Nucleolar number was reduced by MR766 but not PRV. In (b,d) data represent 6 sister cultures from three independent experiments; in (e–g), images of at least 60 randomly selected individual cells with no signs of apoptotic chromatin condensation were analyzed for each condition; such images were collected from two independent experiments; error bars are SEM. Data were analyzed by u-test (b,d) or one-way ANOVA and Tukey’s post-hoc tests (e–g), NS, p > 0.05; *p < 0.05; ***p < 0.001.
Figure 3
Figure 3
Nucleolar localization of the ZIKV capsid protein ZIKV-C. (a) DIV2 rat embryonic cortical neurons were transfected for 48 h with expression vectors for Flag (Fl)-tagged ZIKV-C, its variants, or, other ZIKV proteins as indicated (0.15 μg plasmid DNA/3.5*105 cells); to avoid apoptosis due to possible ZIKV-C-mediated ribosomal stress, an expression plasmid for a dominant negative mutant of Tp53 was also added (Tp53-DD, 0.15 μg plasmid DNA/3.5*105 cells). Representative images of co-immunofluorescence for Flag and the nucleolar marker NPM1 revealed strong perikaryal expression of all ZIKV proteins including predominantly cytosolic localization and nucleolar enrichment of a fraction of Fl-ZIKV-C in many but not all Fl-positive cells (more images of Fl-ZIKV-C immunofluorescence are presented in Supplementary Fig. S6. (b) Quantification of nucleolar enrichment of Fl-ZIKV proteins. To equalize apparent differences in expression efficiency between Fl-ZIKV-C and other constructs, a β-gal expression plasmid was added to the transfections to provide a consistent transfection marker (150 ng plasmid DNA/3.5*105 cells, all other components as described for panel (a), see text for more details). Nucleolar enrichment analysis was performed in β-gal/Fl-double-positive cells. Nucleolar enrichment was observed for Fl-ZIKV-C and, to lesser extent, for the immature, membrane anchored version of Fl-ZIKV-C (C/anch/). Nucleolar enrichment was rare for other constructs including C-terminal deletion mutants of ZIKV-C (C/1-73/ and C/1-82/). Data represent averages of 6 sister cultures from three independent experiments; NS, p > 0.05; *p < 0.05; **p < 0.01 (u-test). (c,d) hNPCs or SH-SY5Y cells were transfected with Fl-ZIKV-C (150 ng plasmid DNA/105 cells) and its localization was analyzed by Fl immunofluorescence 48 h later; nucleolar enrichment was confirmed by co-transfection of a nucleolar/ribosomal marker GFP-RPL4 (hNPCs, 150 ng plasmid DNA/105 cells) or co-immunostaining for NPM1 (SH-SY5Y cells). (e) Neurons were transfected as in (a), and treated with a Pol1-specific inhibitor, BMH21 as indicated. Nucleolar enrichment of both NPM1 and Fl-ZIKV-C was disrupted by BMH21 (nucleolar enrichment of Fl-ZIKV-C was present in 50.6 ± 0.6% or 15.8 ± 5.3% control- or BMH21-treated cells, respectively as determined in two independent experiments).
Figure 4
Figure 4
Disruption of neuronal nucleoli by the overexpressed ZIKV-C. Transfections of ZIKV proteins were as in Fig. 3b with β-gal or empty cloning vector (EV, pBact-16-pl) used as a transfection marker or a negative control, respectively; additional controls included shRNAs targeting Renilla luciferase (shLuc) or the Pol1 co-activator Tif1a (shTif1a, used as a positive control for RS); all analyses were performed 48 h after transfection. (a) Representative images of transfected (i.e. β-gal-positive neurons, arrows) that were co-immunostained for NPM1; 150 ng plasmid DNA of EV or ZIKV-C/3.5*105 cells or 300 ng plasmid DNA of shTif1a/3.5*105 cells were used. Fl-ZIKV-C or shTif1a reduced NPM1 signal in the nucleolus while increasing it in the nucleoplasm (more images are in Supplementary Fig. S7). (b,c) Plasmid DNA dose-dependent reduction of intensity- and territory of the nucleolar NPM1 signal (one-way ANOVA, factor DNA dose, p < 0.001). (d) Fl-ZIKV-C did not affect nucleolar number. (e) Similar reductions of NPM1 signal in the nucleolus after transfections of Fl-ZIKV-C or untagged ZIKV-C. (f–h) Plasmid dosage was as in (a). Nucleolar NPM1 signal was affected by ZIKV-C or shTif1a but not the membrane bound precursor form ZIKV-C(anch) or C-terminal deletion mutants of mature ZIKV-C or other ZIKV proteins (f,g); nucleolar number was reduced only by shTif1a (h). (i–j) In situ run on assay revealed reduction of nascent RNA signal in nucleoli of ZIKV-C-transfected neurons suggesting lower activity of Pol1. Two days after transfections (as in (a)), cells were incubated with 5-ethynyluridine (5-EU) for 2 h, fixed and 5-EU-labelled nascent RNA was detected using Click-It chemistry. Then, β-gal immunofluorescence was performed to identify transfected cells (arrows). In nucleoli of Fl-ZIKV-C-transfected neurons, whole nucleus-normalized accumulation of nascent RNA was moderately reduced (j); however, Pol1 inhibition may be potentially underestimated as average whole nucleus signal was also reduced (Supplementary Table S2). At a low concentration of 33 nM, ActD abolished all nascent RNA signal in nucleoli validating its specificity. Data represent averages of at least 39 cells/condition from two- (b–e,j) or three independent experiments (f–h); NS, p > 0.05; *p < 0.05; ***p < 0.001 (one-way ANOVA and Tukey’s post-hoc tests).
Figure 5
Figure 5
Ribosomal stress-mediated apoptosis of ZIKV-C-overexpressing neurons. DIV2 neurons were transfected as described for Fig. 4 except DN-Tp53 constructs were added only as indicated in (e) and omitted from all other transfections. Unless indicated otherwise, ZIKV protein expression constructs were used at 150 ng plasmid DNA/3.5*105 cells. In (d) besides Fl-ZIKV-C or its EV control, transfections included luciferase reporter constructs (p53-driven firefly luciferase and EF1α-driven Renilla luciferase, 0.1 ng plasmid DNA/105 cells, each; luciferase assay was performed at 24 h post transfection). All analyses except luciferase assays were at 48 h post transfection. (a) Representative images of transfected (i.e. β-gal-positive) neurons. Counterstaining with the DNA dye Hoechst-33258 revealed condensation and fragmentation of nuclear chromatin in ZIKV-C-transfected neurons (arrowheads), a non-apoptotic neuron is pointed by arrows. (b) ZIKV-C induces apoptosis in a plasmid DNA-dose dependent manner (Kruskal-Wallis ANOVA, factor DNA dose, p < 0.05). (c) Similar apoptotic response to ZIKV-C or shTif1a but not ZIKV-C(anch) or ZIKV-C(1–73) or ZIKV-M. (d) ZIKV-C increased Tp53-driven transcription as determined by activity of a co-transfected p53-driven firefly luciferase reporter plasmid. (e,f) As expected for ribosomal stress-mediated apoptosis that involves the RPL11-Tp53 pathway, ZIKV-C-transfected neurons were protected by co-transfection of DN-Tp53 variants, or previously validated shRNAs against Rpl11, or rat Tp53. The shTp53 plasmids also reduced activity of the Tp53-driven luciferase reporter in either vehicle- or nutlin-treated neurons confirming their ability to inhibit Tp53 in that system (Supplementary Fig. S8). Note that as compared to a control cDNA expression vector (pcDNA3.1, (e)) control shRNA (shLuc, (f)), increased baseline apoptosis in non-ZIKV-C-transfected neurons (16.6% or 28.3%, respectively, p < 0.05, u-test). Due to such baseline increases, ZIKV-C was relatively less pro-apoptotic in neurons that received shLuc than pcDNA3.1 (1.3- vs. 1.6 fold, (f) vs. (e)). Data represent averages ± SEM of 4 (b) or 6 (d) sister cultures from two independent experiments, or 6 sister cultures from three independent experiments (c,e,f); NS, p > 0.05; *p < 0.05; **p < 0.01 (u-test).
Figure 6
Figure 6
Capsid protein of Dengue virus (DENV-C) compromises nucleolar integrity and induces neuronal apoptosis. DIV2 neurons or SH-SY5Y cells were transfected with expression plasmids for Flag-(Fl) tagged DENV-C or WNV-C as described for Fig. 4 except DN-Tp53 was omitted from neuronal transfections in panels (j) and (k). (a–c) In neurons and SH-SY5Y cells nucleolar enrichment appeared to be stronger for DENV-C than WNV-C including higher fraction of cells displaying such an enrichment (b), and, higher fluorescence intensity (FI) in the nucleolus (FI quantifications in Supplementary Fig. S10). (d–g) In β-gal-positive neurons that were co-transfected with expression plasmids for β-gal and flaviviral Cs, DENV-C but not WNV-C reduced nucleoplasm-normalized NPM1 signal intensity in the nucleolus and NPM1-positive nucleolar territory (arrowheads in (d), black bars in (e,f)); arrows point a Fl-WNV-C overexpressing neuron. However, number of NPM1-positive nucleoli was unaffected (g). (h,i) In situ run on assay revealed that whole nucleus-normalized nucleolar accumulation of nascent RNA was reduced in DENV-C but not WNV-C-transfected neurons. In both cases, nascent RNA signal was also reduced in whole nuclei suggesting inhibition of extranucleolar transcription and/or lower uptake of 5-EU into cells (Supplementary Table S2). Hence, anti-Pol1 effects of DENV-C may be potentially underestimated. (j) Increased neuronal apoptosis in response to DENV-C- but not WNV-C. (k) Tp53 reporter assay was performed as for Fig. 5d. DENV-C and WNV-C activated Tp53-driven transcription; the activation was stronger with the RS-inducing DENV-C than WNV-C. Data represent averages ± SEM of 6- (b,j) or 9 sister cultures (k) from three independent experiments, or, at least 51 cells- from three independent experiments (e–g,i); NS, p > 0.05; *p < 0.05; **p < 0.01, ***p < 0.001 (u-test in (b,j,k); one-way ANOVA with Tukey’s posthoc tests in (e–g,i)).
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