Fig. 2.

The cytopathic effects of ZIKV proteins. (A) The effects on cell proliferation. (a) Expression of ZIKV mRNA transcripts measured 24 h after GI by RT-PCR. The order of proteins from 1–14: anaC, C, PR, M, prM, E, 2K, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. M, molecular marker. (b) Effect of ZIKV expression on fission yeast colony formation. Agar plates are numbered as in a. An empty pYZ1N vector was used as a control and designated as zero. ZIKV-carrying cells grown on selective EMM agar plates were incubated at 30 °C for 4–5 d before the images were captured. Off, gene-suppressed; On, gene-induced. (c) Growth curve analysis was used to quantify growth inhibition by ZIKV proteins. Only the effect of ZIKV proteins (anaC, C, E, prM, M, NS2B, and NS4A) that affected yeast colony-forming abilities as demonstrated in b are shown. Cell growth was measured spectrophotometrically at OD₆₅₀ over the indicated time period. The experiment was repeated at least three times, and the SEs of each time point were calculated. ●, gene-on, the ZIKV gene was induced; ○, gene-off, the ZIKV gene was suppressed. (B) The effects on cell morphology. Only ZIKV proteins that affected cell proliferation as demonstrated in A, c are shown. The effects of all ZIKV proteins on fission yeast nuclear morphology are included in Fig. S3. (a) Individual cell morphology. Each image was taken 45 h after GI using bright-field microscopy. Note cell hypertrophy (arrow) in the NS4A-expressing cells. Vec, empty vector. (b) Overall cell morphology as shown by the FSC analysis. Ten thousand cells were measured 48 h after GI. The FSC measures the distribution of all cell sizes. The SSC determines intracellular complexity. (C) Effects on nuclear morphology and cell-cycle regulation. (a) Nuclear morphology. Cells were stained with Hoechst blue fluorescent DNA dye. All cell and nuclear morphologies were examined 45 h after GI. (Scale bar, 10 μm.) Effects of all ZIKV proteins on fission yeast nuclear morphology are included in Fig. S4. (b) The effect of ZIKV protein on cell-cycle G1 regulation. Cells were grown in regular EMM medium in which cells normally reside in the G2/M phase of the cell cycle. (c) Effect of ZIKV proteins on cell-cycle G2/M regulation. Cells were grown in a low-nitrogen EMM medium that enriches cells in the G1 phase of the cell cycle, as described previously (1, 2). Cell-cycle profiles were measured by DNA content using flow cytometric analysis 48 h after GI. Average and SD values were calculated based on the results of three independent experiments. A pairwise Student t test was conducted to compare the DNA content values of each ZIKV protein with and without GI. *P < 0.01. Arrows indicate the location of differences (C, b and c) or where the abnormal cells reside (B, b and C, a).