Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth

Abstract

The suspected link between infection by Zika virus (ZIKV), a re-emerging flavivirus, and microcephaly is an urgent global health concern. The direct target cells of ZIKV in the developing human fetus are not clear. Here we show that a strain of the ZIKV, MR766, serially passaged in monkey and mosquito cells efficiently infects human neural progenitor cells (hNPCs) derived from induced pluripotent stem cells. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection increases cell death and dysregulates cell-cycle progression, resulting in attenuated hNPC growth. Global gene expression analysis of infected hNPCs reveals transcriptional dysregulation, notably of cell-cycle-related pathways. Our results identify hNPCs as a direct ZIKV target. In addition, we establish a tractable experimental model system to investigate the impact and mechanism of ZIKV on human brain development and provide a platform to screen therapeutic compounds.

Figure 1. ZIKV Infects hiPSC-derived Neural Progenitor Cells with High Efficiency

(A–B) Sample confocal images of forebrain-specific hNPCs (A) and immature neurons (B) 56 hours after infection with ZIKV supernatant, and immunostained for ZIKV envelop protein (ZIKVE; green) and DAPI (gray). Cells were differentiated from the C1-2 hiPSC line. Scale bars: 20 μm. (C) Quantification of infection efficiency for different cell types, including hESCs, hiPSCs, hNPCS derived from two different hiPSCs, and immature neurons 1 or 9 days after differentiation from hNPCs. Both hESCs and hiPSCs were analyzed 72 hours after infection, whereas all other cells were analyzed 56 hours after infection. Numbers associated with bar graphs indicate numbers of independent experiments. Values represent mean ± SD (*P < 0.01; Student’s t-test) (D) Production of infectious ZIKV particles by infected hNPCs. Supernatant from hNPC cultures 72 hours after ZIKV infection was collected and added to Vero cells for 2 hours. The Vero cells were further cultured for 48 hours. Shown are sample images of ZIKVE immunostaining (green) and DAPI (gray). Scale bars: 20 μm. See also Figure S1 and Table S1

Figure 2. ZIKV-infected hNPCs Exhibit Increased Cell Death and Dysregulated Cell Cycle Progression and Gene Expression

(A–B) Increased cell death of ZIKV-infected hNPCs. Shown in (A) are sample images of immunostaining of hNPCs for ZIKVE (green) and cleaved-caspase 3 (Cas3; red) and DAPI (gray) 72 hours after ZIKV infection. Scale bars: 20 μm. Shown in (B) is the quantification. Values represent mean ± SEM (n = 6; *P < 0.01; Student’s t-test). (C) Cell cycle perturbation of hNPCs infected by ZIKV. Shown are sample flow cytometry analyses of distributions of hNPCs (from the C1-2 line) at different phases of cell cycle 72 hours after ZIKV or Mock infection. For the mixture sample, mock and infected hNPCs were mixed at a ratio of 1:1 following Propidium Iodide staining of each sample. (D–E) RNA-seq analysis of hNPCs (C1-2 line) 56 hours after ZIKV or mock infection. Genes with significant differences in expression between infected and uninfected hNPCs were subjected to GO analyses. The top 10 most significant terms are shown for downregulated (D) and upregulated (E) genes, respectively. The −log10 p-values are indicated by bar plots. An additional term of regulation of programmed cell death is also shown for upregulated genes (E). See also Figure S2 and Table S2