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).
