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).