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.