Zika virus infection dysregulates human neural stem cell growth and inhibits differentiation into neuroprogenitor cells

Abstract

The current outbreak of Zika virus-associated diseases in South America and its threat to spread to other parts of the world has emerged as a global health emergency. A strong link between Zika virus and microcephaly exists, and the potential mechanisms associated with microcephaly are under intense investigation. In this study, we evaluated the effect of Zika virus infection of Asian and African lineages (PRVABC59 and MR766) in human neural stem cells (hNSCs). These two Zika virus strains displayed distinct infection pattern and growth rates in hNSCs. Zika virus MR766 strain increased serine 139 phosphorylation of histone H2AX (γH2AX), a known early cellular response proteins to DNA damage. On the other hand, PRVABC59 strain upregulated serine 15 phosphorylation of p53, p21 and PUMA expression. MR766-infected cells displayed poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Interestingly, infection of hNSCs by both strains of Zika virus for 24 h, followed by incubation in astrocyte differentiation medium, induced rounding and cell death. However, astrocytes generated from hNSCs by incubation in differentiation medium when infected with Zika virus displayed minimal cytopathic effect at an early time point. Infected hNSCs incubated in astrocyte differentiating medium displayed PARP cleavage within 24-36 h. Together, these results showed that two distinct strains of Zika virus potentiate hNSC growth inhibition by different mechanisms, but both viruses strongly induce death in early differentiating neuroprogenitor cells even at a very low multiplicity of infection. Our observations demonstrate further mechanistic insights for impaired neuronal homeostasis during active Zika virus infection.

Figure 1

Subcellular localization of Zika virus E glycoprotein in virus-infected cells. Vero cells infected with PRVABC59 and MR766 displayed similar pattern of perinuclear and nuclear immunofluorescence of the E glycoprotein (b and c). Although hNSCs infected with PRVABC59 displayed localization of E glycoprotein primarily as perinuclear punctate dots, unlike MR766 (e and f). Mock-infected control (a,d) and virus-infected cell nuclei were stained with DAPI

Figure 2

γH2AX expression as DDR is significantly increased in MR766-infected hNSCs as compared with PRVABC59-infected cells. Western blot analysis for γH2AX protein expression from Zika virus-infected hNSCs is shown (a). Blots were reprobed with total H2AX and actin-specific antibodies. Results of densitometric analysis are presented on the right. *P < 0.05, **P < 0.001. Confocal microscopy images from both virus-infected cells displaying localization of γH2AX (red) and virus E protein (green) in merged image panels are shown (b). The nuclei were stained with DAPI (blue). Nuclear localization of distinct dots of γH2AX is shown. hNSCs infected with MR766 also displayed higher expression of γH2AX in nucleus with apoptotic nuclei (right panel)

Figure 3

Zika virus-infected hNSCs display DDR response and promotes expression of cell cycle arrest and apoptotic markers. Western blot analysis for phospho-p53, p53, p21 and PUMA in PRVABC59 or MR766-infected hNSCs using specific antibodies (a and b). The blot was reprobed with an antibody to actin as an internal control. Densitometry analyses was performed from three experiments using ImageJ software and shown on the right. Data are represented as mean±S.D. Small bar indicates S.E. (*P<0.05; **P<0.01). PRVABC59 or MR766-infected hNSCs lysates were subjected to western blot analysis for PARP and caspase-3 using specific antibodies (c and d). The blot was reprobed with an antibody to actin as an internal control

Figure 4

Cytopathic role of Zika virus infection in early differentiating neuroprogenitor cells. Mock-infected control, PRVABC59 and MR766-infected hNSCs were treated 24 h after infection with astrocyte differentiating medium for generation of neuroprogenitor cells. Cell viability assay of hNSCs infected with PRVABC59 and MR766 followed by incubation in differentiating medium for astrocytes are shown (a). Calcein AM stained live cells (green) and ethidium homodimer stained dead cells (red) are shown. A quantitation of live cells is shown on the right. Representative images of cytopathic effect after 48 h of incubation in differentiating medium from four independent experiments are shown (b). Western blot analysis was performed for analysis of PARP cleavage from Zika virus-infected cell lysates using specific antibody (c). The blot was reprobed with an antibody to actin as an internal control. Densitometry analyses was performed from three experiments using ImageJ software and shown on the right. Mock-infected or Zika virus-infected hNSCs incubated in differentiating medium for astrocyte generation were separately stained for GFAP marker (red) and viral E envelope glycoprotein (green) are shown (d), nuclei were stained with DAPI (blue color). We could not determine GFAP expression in PRVABC59-infected hNSCs for faster detachment from culture plate. Western blot analysis of GFAP was performed from mock, PRVABC59 or MR766-infected cell lysates using specific antibody (e). The blot was reprobed with an antibody to actin as an internal control. Data are represented as mean±SD. Small bar indicates standard error (*P<0.05, **P < 0.001)

Figure 5

Summary of observations on neuronal damage by two different strains of Zika virus. hNSCs infected with PRVABC59 induced activation of the p53-p21 signaling axis, driving cells toward cell cycle arrest, whereas MR766-infected hNSCs were driven by apoptotic pathway. These cells infected with both Zika virus strains followed by incubation in astrocyte differentiating medium displayed cell death