The Zika virus (ZIKV) is related to flaviviruses that cause dengue and yellow fever, and is spread via mosquito (Ae. aegypti and Ae. albopictus) bites and secondarily via sexual transmission. ZIKV infection in adults is associated with mild dengue-like disease or Guillain-Barré syndrome, an autoimmune disorder of the peripheral nervous system. Most alarmingly, ZIKV is also transmitted across the placental barrier during pregnancy and can lead to the severe birth defect microencephaly (underdevelopment of the brain).
Current research efforts aim to understand the molecular basis of ZIKV infection and pathology and uncover information that could help in the development of therapeutics and/or a vaccine. For this edition of the journal club, we will discuss a study led by researchers at USC and published in Cell Stem Cell demonstrating ZIKV activates autophagy and inhibits neurogenesis in neural stem cells via inhibition of the Akt-mTOR signaling pathway. The authors used neurospheres derived from neuronal stem cells (NSCs), complementing studies performed in mouse and macaque models to shed light on the mechanism of ZIKV neuropathology.
Neurogenesis is a crucial process in the developing brain involving differentiation of NSCs into neurons, and is regulated by the Akt/mTOR signaling pathway, among others. Up-regulation of Akt or mTOR is genetically associated with megaencephaly while down-regulation is associated with microencephaly. mTOR signaling also regulates cellular autophagy; mTOR activation by Akt suppresses autophagy while inactivation by AMPK/p53 activates autophagy. Different viruses either activate or repress cellular autophagy; recent studies report ZIKV and dengue (DENV) hijack cellular autophagy during production of new viral particles on the endoplasmic reticulum. Knowing this, Liang et al investigated whether ZIKV-induced autophagy could influence neurogenesis.
To characterize ZIKV effects on growth, differentiation and cell death in developing neuroepithelia, Liang et al first assayed in vitro neurosphere formation. Neurospheres infected with ZIKV for 3 days were smaller and had increased cell death and decreased proliferation compared to mock-infected controls. Activation of autophagy was elevated by ZIKV infection in NSCs, HeLa, and MEF cells. Turning up autophagy with rapamycin increased ZIKV viral titer, while dampening autophagy with 3-MA or chloroquine decreased ZIKV titer, confirming the association of ZIKV replication and cellular autophagy.
To establish which ZIKV products were responsible for effects on neurogenesis, the investigators screened all ten ZIKV proteins using lentiviral delivery. The nonstructural proteins NS4A and NS4B, either singly or together, decreased the size of neurospheres. This is specific for ZIKV, as expression of DENV NS4A/NS4B did not have an effect. Furthermore, ZIKV NS4A and NS4B proteins reduced proliferation and differentiation of dissociated NSCs into neurons and astrocytes, as indicated by immunofluorescent (IF) analysis of b3-tubulin and GFAP markers, respectively. Expression of NS4A and/or NS4B was also sufficient to induce autophagy, as shown by appearance of GFP-LC3 puncta in HeLa cells and LC3-II:LC3-I ratiometric densitometry in HeLa and NSCs.
Finally, Akt/mTOR activation was evaluated by western blotting with phospho-specific antibodies in the presence or absence of ZIKV. Akt activity is regulated by phosphorylation at residues T308 and S473, and active Akt phosphorylates mTOR on S2448. All three phospho-specific antibodies indicated time-dependent inhibition of Akt/mTOR after ZIKV infection of NSCs. Lentivirus-mediated expression of NS4A or NS4B, but not of other ZIKV proteins, was sufficient to inhibit activation of Akt and mTOR in HeLa, and NS4A or NS4B were also able to reduce activation of Akt/mTOR in serum-starved NSCs or HeLa in response to insulin. These data support the conclusion that these two nonstructural ZIKV proteins interfere with normal Akt/mTOR activation in the host cell in order to activate autophagy, contributing to loss of neurons and Zika neuropathology.
Future studies using structural approaches might reveal further details about the mechanism of Akt inhibition and how it relates to Zika virus life cycle. Therapeutic strategies to alleviate neuropathology after Zika infection would likely be difficult to implement during pregnancy. However, it could be worthwhile to investigate whether the Akt pathway is also targeted by ZIKV in blood cells, endothelia, epidermis or other tissues, as these are more therapeutically accessible and may be the initial site(s) of Zika colonization in humans. Much work remains to be done.
Interested in learning more about PI3K/Akt signaling? Download the PI3K/Akt Pathway Handout.