The outbreak of Zika virus (ZIKV) reported in Brazil earlier this year is associated with higher incidence of babies born with microcephaly. Liang et al. (2016) recently presented evidence of ZIKV protein disruption of neuronal signaling pathways, which inhibits development in fetal neuronal stem cells (fNSCs), showing both the molecular basis behind infection and indicating potential therapeutic targets.1
Although various research groups have shown strong evidence to support the role of ZIKV in microcephaly, the molecular pathways by which this happens are less well known. In this study, Liang and coworkers infected fNSC cultures with three strains of ZIKV (MR766, 1bH30656 and H/PF/2013) to examine in vitro pathogenesis. The team concentrated on the Akt-mTOR signaling pathway in these cells, since it is an important factor in normal brain development during the prenatal period and is implicated in other developmental anomalies in the central nervous system.
The researchers used fNSCs collected from second-trimester fetuses (18–22 weeks post-conception), choosing this period because it reflects when the developing baby is most susceptible to the effects of ZIKV infection. Following extraction, they established stable cultures of the fNSCs before infecting them with one of the three strains of ZIKV obtained. After infection, the team used a number of standard laboratory techniques to characterize the effect of ZIKV infection on the cells. These included measuring induced cell death using the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Scientific) and proliferation, in addition to following markers of neuronal development and stem cell transformation such as neurosphere formation. They also examined cellular autophagy as a marker of viral influence on host cellular processes and characterized protein changes by immunoblotting through an ECL approach (Thermo Scientific).
Following in vitro infection, the researchers found increased cell death with a reduction in proliferation and neurosphere formation with each of the three strains of ZIKV. Looking at markers of autophagy, the team saw an increase with each viral infection. When they examined the same markers in autophagy knockout cells, they found the deletion coincided with a reduced burden of ZIKV infection.
Next, Liang et al. narrowed down the roles of the 10 ZIKV-associated proteins in this disruption. Using lentivirus transduction of fNSCs, the team examined each protein in isolation until they could demonstrate involvement of the two proteins responsible: NS4A and NS4B. The fNSCs expressing these two ZIKV proteins showed reduced neurosphere formation; with cells co-expressing both ZIKV proteins, the effect was even more marked, with a reduction in the size of neurospheres and inhibition of differentiation. The team could not replicate these findings by using NS4A and –B proteins from a closely related flavivirus, Dengue virus, showing that the effect is specific to ZIKV infection.
Liang et al. also found that ZIKV NS4A and NS4B coexpression induced autophagy in the fNSC cells. Moreover, they could replicate this result in a transiently transfected HeLa cell preparation. This increase in autophagy coincided with enhanced ZIKV replication, thus supporting previous studies that showed viral control of cellular pathways to enhance infection. Treating cells with the autophagy inducer rapamycin also promoted ZIKV infectivity and replication.
Liang et al. then turned to examining cellular signaling pathway phosphorylation, concentrating on Akt, which activates through Thr308 and Ser473. Both ZIKV infection and NS4A and NS4B expression in vitro reduced Akt phosphorylation at these two sites. This in turn decreased mTOR phosphorylation and increased autophagy.
The authors acknowledge that the three strains used in the experimental process presented in their paper might not fully represent the ZIKV outbreak in Brazil. However, since the two proteins implicated—NS4A and NS4B—are closely conserved among ZIKV strains, the team is confident that the cellular pathway disruptions characterized fully reflect the molecular pathogenesis of the infection in fNSCs. Furthermore, Liang et al. suggest that the findings showing Akt-mTOR pathway inhibition represent potential therapeutic targets for further investigation.
1. Liang, Q., et al. (2016) “Zika virus NS4A and NS4B proteins deregulate Akt-mTOR signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy,” Cell Stem Cell, 19 (pp.1–9). doi: 10.1016/j.stem.2016.07.019.
Post Author: Amanda Maxwell. Amanda is a freelance science writer and digital space explorer with a passionate curiosity for science and technology. She enjoys translating complex theories and subjects creatively into everyday language for all audiences. Equipped with a bachelor’s degree in veterinary medicine and a PhD in protein chemistry/small animal critical care nutrition, she brings clinical experience and practical research oversight into her writing.