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Short Article
Zika Virus NS4A and NS4B
Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cells toInhibit Neurogenesis and Induce AutophagyGraphical Abstract
Highlights
d ZIKV infects human fNSCs, leading to defective neurogenesis
and increased autophagy
d Expression of ZIKV NS4A andNS4B blocks neurogenesis and
promotes autophagy
d Two ZIKV proteins, NS4A and NS4B, inhibit Akt-mTOR
signaling
Liang et al., 2016, Cell Stem Cell 19, 1–9November 3, 2016 ª 2016 Elsevier Inc.http://dx.doi.org/10.1016/j.stem.2016.07.019
Authors
Qiming Liang, Zhifei Luo,
Jianxiong Zeng, ...,
Berislav V. Zlokovic, Zhen Zhao,
Jae U. Jung
[email protected] (Q.L.),[email protected] (Z.Z.),[email protected] (J.U.J.)
In Brief
Liang et al. show that after infection of
human fetal neural stem cells, the ZIKV
proteins NS4A and NS4B inhibit the Akt-
mTOR signaling pathway, disrupting
neurogenesis and inducing autophagy.
Their study therefore identifies candidate
molecular determinants of ZIKV
pathogenesis and highlights potential
targets for therapeutic intervention.
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
Cell Stem Cell
Short Article
Zika Virus NS4A and NS4B Proteins DeregulateAkt-mTOR Signaling in Human Fetal Neural StemCells to Inhibit Neurogenesis and Induce AutophagyQiming Liang,1,4,* Zhifei Luo,2,3 Jianxiong Zeng,1 Weiqiang Chen,1 Suan-Sin Foo,1 Shin-Ae Lee,1 Jianning Ge,1
Su Wang,5,6,7 Steven A. Goldman,5,6,7 Berislav V. Zlokovic,2,3 Zhen Zhao,2,3,* and Jae U. Jung1,8,*1Department of Molecular Microbiology and Immunology2Department of Physiology and Biophysics3Zilkha Neurogenetic Institute
Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA4Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine,
Shanghai, 200025, China5Center for Translational Neuromedicine6Department of Neurology
University of Rochester, Rochester, NY 14642, USA7Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark8Lead Contact
*Correspondence: [email protected] (Q.L.), [email protected] (Z.Z.), [email protected] (J.U.J.)
http://dx.doi.org/10.1016/j.stem.2016.07.019
SUMMARY
The current widespread outbreak of Zika virus (ZIKV)infection has been linked to severe clinical birth de-fects, particularly microcephaly, warranting urgentstudy of the molecular mechanisms underlyingZIKV pathogenesis. Akt-mTOR signaling is one ofthe key cellular pathways essential for brain develop-ment and autophagy regulation. Here, we show thatZIKV infection of human fetal neural stem cells(fNSCs) causes inhibition of the Akt-mTOR pathway,leading to defective neurogenesis and aberrant acti-vation of autophagy. By screening the three struc-tural proteins and seven nonstructural proteins pre-sent in ZIKV, we found that two, NS4A and NS4B,cooperatively suppress the Akt-mTOR pathway andlead to cellular dysregulation. Corresponding pro-teins from the closely related dengue virus do nothave the same effect on neurogenesis. Thus, ourstudy highlights ZIKV NS4A and NS4B as candidatedeterminants of viral pathogenesis and identifies amechanism of action for their effects, suggesting po-tential targets for anti-ZIKV therapeutic intervention.
INTRODUCTION
Zika virus (ZIKV), a reemerging arthropod-bone flavivirus, was
initially isolated from Rhesus macaque in Uganda as early as
1947, while the first human infection was reported in 1954
(Dick, 1952; Dick et al., 1952; Simpson, 1964). Although sexually
transmitted cases have been recently documented, ZIKV is most
commonly transmitted through the bites of infected Aedes
mosquitoes (Musso et al., 2015; Venturi et al., 2016). Individuals
infected by ZIKV typically develop a mild or unapparent dengue-
like disease (Duffy et al., 2009). However, mounting evidence has
linked ZIKV infection to neurological defects in newborns (De
Carvalho et al., 2016). Furthermore, ZIKV was detected in the
amniotic fluids of pregnant women as well as in the brain tissues
of microcephalic fetuses, suggesting that ZIKV can potentially
cross the placental barrier to infect fetuses (Calvet et al., 2016;
Mlakar et al., 2016). In addition, an increased rate of Guillain-
Barre syndrome was noted following the ZIKV outbreak in
French Polynesia from 2013 to 2014 (Cauchemez et al., 2016).
The World Health Organization declared a Public Health Emer-
gency of International Concern (Heymann et al., 2016), and the
Centers for Disease Control and Prevention confirmed that
ZIKV causes microcephaly and other birth defects. Although
ZIKV infection impairs the growth of neurospheres and brain or-
ganoids derived from iPSCs (Garcez et al., 2016; Qian et al.,
2016), the molecular mechanism by which ZIKV infection in-
duces fetal microcephaly remains elusive. In particular, dengue
virus (DENV), a closely related member of the flaviviridae family,
has not been linked to either microcephaly or defects in neuro-
genesis (Garcez et al., 2016), suggesting that ZIKV’s neuropa-
thology might be causally linked to those differences in its
sequence from dengue.
Neurogenesis, the key process by which neurons are differen-
tiated from neural stem cells (NSCs) or neural progenitor cells
(NPCs), is most active during prenatal development and respon-
sible for populating the growing brain with neurons (Gotz
and Huttner, 2005). Genetic defects that are associated with
neurogenesis and migration often result in human develop-
mental neurological disorders including microcephaly (Ming
and Song, 2011). A number of key cellular signaling pathways,
including the PI3K-Akt-mTOR pathway, are essential for neuro-
genesis from NSCs, as well as for subsequent migration and
maturation (Lee, 2015; Wahane et al., 2014). Recent studies
have shown that activating mutations in the PI3K-Akt-mTOR
pathwaymay occur in brain overdevelopment syndromes, which
Cell Stem Cell 19, 1–9, November 3, 2016 ª 2016 Elsevier Inc. 1
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Figure 1. ZIKV Infection Impairs Neurosphere Formation and Elevates Autophagy in fNSCs
(A) Representative images from Live/Dead cell viability assay in cultured fNSCs at 5 dpi with three strains of ZIKV (MR766, IbH30656, or H/PF/2013) or mock
treatment. Images were taken using live cell imaging. Scale bar, 20 mm.
(B) Quantification of percentage of cell death as described in (A). Mean ± SEM; p < 0.05 by one-way ANOVA.
(C) Representative images showing neurosphere formation at 3 dpi with three strains of ZIKV or mock treatment. Scale bar, 100 mm.
(D and E) Quantification of number of neurospheres formed per 1 3 105 fNSCs (D) and neurosphere size by diameter measurement (E) in conditions as in (C).
Mean ± SEM; p < 0.05 by one-way ANOVA.
(legend continued on next page)
2 Cell Stem Cell 19, 1–9, November 3, 2016
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
includemegalencephaly-capillary malformation andmegalence-
phaly-polydactyly-polymicrogyria-hydrocephalus (Mirzaa et al.,
2013). In contrast, mTOR inhibition in the developing brain
causes microcephaly (Cloetta et al., 2013). Upstream to
mTOR, Akt is the central signaling molecule in the PI3K pathway,
and it plays critical roles in brain development as well as synaptic
plasticity (Franke, 2008). Non-functional Akt mutation leads to
microcephaly in humans (Mirzaa et al., 2013), while activating
Akt mutation causes megalencephaly (Nellist et al., 2015; Riviere
et al., 2012). Expression of dominant-negative Akt also blocks
neuronal production from human NSCs isolated from fetuses
in vitro (Guo et al., 2013). Hence, human pathogens, including
DNA viruses, have been found to hijack the PI3K-Akt-mTOR
pathway for their successful replication in mammalian cells
(Buchkovich et al., 2008). Yet despite the common outcomes
of ZIKV infection and PI3K-Akt-mTOR pathway inhibition, no
causal association between the two has yet been reported.
Flaviviruses, such as DENV, Hepatitis C virus (HCV), and ZIKV,
have been shown to modulate cellular autophagy to benefit their
replication in host cells (Hamel et al., 2015; Heaton and Randall,
2010; Sir et al., 2012). Macroautophagy, often referred to as
autophagy, is an important catabolic process involving the for-
mation of double-membrane vesicles, called autophagosomes,
which sequester cytoplasmic damaged organelles, protein ag-
gregates, or invading intracellular pathogens for degradation
(Levine et al., 2011). The mTOR kinase serves as a gatekeeper
for autophagy induction: the activation of mTOR by Akt and
MAPK signaling suppresses autophagy, and the inactivation of
mTOR by AMPK and p53 signaling promotes autophagy. Auto-
phagy is also an active cellular protective mechanism against
viral infection; viruses thus try to modulate autophagy to benefit
their life cycles. Certain herpesviruses suppress autophagy by
their viral proteins to establish persistent infection (Liang et al.,
2013, 2015; Williams and Taylor, 2012). Interestingly, certain fla-
viviruses such as ZIKV and DENV have recently been reported to
hijack autophagy to similarly support viral replication (Hamel
et al., 2015; Heaton and Randall, 2010). Specifically, DENV
NS4A upregulates autophagy in epithelial cells (McLean et al.,
2011). However, it is currently unknown whether ZIKV infection
induces autophagy through a similar mechanism to DENV.
Nevertheless, understanding the molecular mechanism underly-
ing ZIKV-induced autophagy activation in host cells may shed
insight on its pathogenesis.
Recent reports using human NSCs have demonstrated the
vulnerability of these cells to ZIKV infection (Garcez et al.,
2016; Tang et al., 2016), as well as the growth defects of iPSC-
derived neural organoids in response to ZIKV infection (Garcez
et al., 2016; Qian et al., 2016). Several groups also defined the
birth defects from different pregnant mouse models when
exposed to ZIKV (Cugola et al., 2016; Lazear et al., 2016; Li
(F and G) Representative confocal images showing 3D reconstruction of neurosp
fNSC-specific markers; ZIKV was immunostained against its E protein in (F); apo
(H) ZIKV infection induces autophagy in fNSCs. fNSCs were infected with ZIKV a
(I) fNSCs infected with ZIKV at MOI 0.1 were fixed and stained with indicated a
ANOVA.
(J) Autophagy is required for the efficient replication of ZIKV. fNSCs were infecte
drugs (rapamycin 50 nM, 3-MA 2 mM, chloroquine 5 mM) at 1 hpi. The mRNA lev
See also Figures S1 and S2.
et al., 2016; Miner et al., 2016; Rossi et al., 2016). These suggest
the direct cause of ZIKV for human microcephaly via intrauterine
infection and defective neurogenesis. However, no analogous
studies have been performed on native human fetal tissues or
NSCs, largely due to the limited availability of fetal human tissue.
Here, we utilized two primary isolates of fNSCs, recovered
from second trimester human fetuses, a gestational period of
great ZIKV vulnerability in human brain development, to study
how ZIKV infection impairs fetal brain development. We found
that ZIKV infection of human fNSCs results in the inhibition of
neurosphere growth and neurogenic differentiation potential,
as well as the induction of autophagy. Further screening for
ZIKV proteins revealed that the cooperation of NS4A and
NS4B strongly suppresses host Akt-mTOR signaling, potentially
leading to the impairment of neurogenesis of human fNSCs and
the upregulation of autophagy, synergistically promoting viral
replication.
RESULTS
Infection of Human fNSCs with ZIKV Leads to ImpairedNeurosphere Formation and Elevated AutophagyIn order to model ZIKV infection in human fNSCs (Guo et al.,
2013; Keyoung et al., 2001; Wang et al., 2010), we first infected
fNSCs with three ZIKV strains (MR766, IbH30656, and H/PF/
2013) at a multiplicity of infection (MOI) of 0.01. Consistent with
NSCs derived from human iPSCs (Garcez et al., 2016; Tang
et al., 2016), ZIKV efficiently infected fNSCs (Figures S1A–
S1C), and the infection of fNSCs with ZIKV MR766, IbH30656,
and H/PF/2013 led to 4.4-, 5.2-, and 5.5-fold increases of cell
death, respectively, when compared with mock treatment (Fig-
ures 1A and 1B). At 3 days post-infection (dpi), mock-treated
fNSCs in suspension culture formed neurospheres that had an
average size of 81.7 mm (Figures 1C–1E). However, MR766-,
IbH30656-, or H/PF/2013-infected fNSCs at MOI 0.01 formed
fewer neurospheres with smaller average sizes of 54.3 mm,
51.2 mm, or 52.6 mm, respectively (Figures 1D and 1E). Immuno-
histological analysis on 7 dpi neurospheres showed the pres-
ence of ZIKV E antigen within the neurospheres (Figures 1F
and S1D). In addition, ZIKV-infected neurospheres contained
more apoptotic cells than mock-infected neurospheres, as
shown by in situ terminal deoxynucleotidyl transferase-mediated
digoxigenin-dUTP nick-end labeling (TUNEL) (Figure 1G). Corre-
lation analysis between neurosphere sizes and cell death from 30
mock-treated and 30 ZIKV-infected neurospheres showed that
ZIKV infection augmented the death of fNSCs in the neuro-
spheres, in proportion to their size (Figure S1E).While ZIKV infec-
tion did not alter the expression of neural stem cell markers,
e.g., Nestin and SOX2 (Figures 1F and 1G), it led to the progres-
sive reduction of fNSC proliferation as reflected by BrdU
heres at 7 dpi with MR766 or mock treatment. Nestin and SOX2 were used as
ptotic cell death was marked by TUNEL staining in (G). Scale bar, 50 mm.
nd LC3 processing was examined by immunoblot at indicated time points.
ntibodies and LC3 puncta were counted. Mean ± SEM; p < 0.05 by one-way
d with ZIKV MR766 at MOI 0.5, and the medium was changed with indicated
els of ZIKV were measured by RT-qPCR at 10 hpi.
Cell Stem Cell 19, 1–9, November 3, 2016 3
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
incorporation (Figure S1F). These results show that ZIKV infec-
tion not only induces the death of human fNSCs but also impairs
their proliferation and clonal expansion in neurospheres.
The endoplasmic reticulum provides a membrane platform for
the biogenesis of flavivirus replication complex, and autophagy-
dependent processing of lipid droplets is required for efficient
flavivirus replication (Heaton and Randall, 2010). To determine
changes in the level of autophagy upon ZIKV infection, we exam-
ined the light chain 3 (LC3)-I to LC3-II conversion and the forma-
tion of LC3 punctate structure in ZIKV-infected fNSCs. Infection
with ZIKV strains MR766 and IbH30656 efficiently induced LC3-I
to LC3-II conversion and LC3 puncta formation of fNSCs in the
presence or absence of lysosome inhibitor bafilomycin A1 (Fig-
ures 1H–1I and S1G–S1I). The p62/SQSTM level also decreased
due to ZIKV-mediated autophagosome maturation (Figures 1H
and S1G). Similar results were obtained from HeLa cells and
MEFs (Figures S1J–S1L and S2A–S2D). To investigate the role
of autophagy in ZIKV infection, we assessed ZIKV replication us-
ing Atg3 knockout (KO) MEFs, in which autophagy is completely
defective due to the loss of the Atg3 E2 enzyme. ZIKV replication
was reduced by approximately 7-fold in Atg3 KO MEFs
compared to wild-type MEFs (Figures S2E and S2F). In addition,
ZIKV-infected Atg3 KO MEFs showed no detectable LC3-I to
LC3-II conversion (Figure S1L). Similar results were obtained in
Atg5 KO MEFs, and Atg3 or Atg13 knockdown fNSCs (Figures
S2G–S2I). Accordingly, the induction of autophagy by rapamycin
promoted ZIKV load in both fNSCs and HeLa cells, whereas the
inhibition of autophagy by 3-MA or chloroquine impaired ZIKV
load (Figures 1J, S2J, and S2K). These results indicate that
ZIKV infection induces autophagy in fNSCs, which in turn leads
to increased ZIKV replication and viral load.
ZIKV NS4A and NS4B Suppress Neurogenesis of HumanfNSCsPrevious reports (Garcez et al., 2016; Qian et al., 2016) as well as
our current study demonstrate that ZIKV infection impairs the
growth and proliferation of iPSC-derived NSCs and fNSCs.
Like other flavivirus family members, ZIKV is expected to encode
ten viral proteins including three structural and seven non-struc-
tural proteins (NSs). To determine which viral proteins might play
inhibitory roles in cell proliferation and neurosphere formation,
human fNSCs were transduced with lentivirus containing each
ZIKV gene and examined for neurosphere formation (Figures
S3A–S3C). Expression of each ZIKV protein was detected by
immunoblotting at 2 dpi (Figure S3D), and the size and number
of neurospheres were measured at 7 dpi. Interestingly, human
fNSCs expressing NS4A or NS4B exhibited impaired neuro-
sphere formation (Figure 2A), as reflected in their reduced effi-
ciency of neurosphere production from standard aliquots of
1 3 105 fNSCs (Figure 2B). Moreover, the majority of neuro-
spheres (>90%) were less than 100 mm in diameter (Figure 2C),
and the average neurosphere size significantly reduced by
40.3% or 32.3%, respectively, when NS4A or NS4B was ex-
pressed (Figure S3C). Remarkably, co-expression of NS4A and
NS4B resulted in further inhibition of neurosphere formation,
with the near absence of neurospheres with diameters of
100 mm or higher and a reduction of average neurosphere size
by 52.0% (Figures 2A–2C and S3A–S3C). Unlike ZIKV NS4A
and NS4B, co-expression of DENV NS4A and NS4B did not
4 Cell Stem Cell 19, 1–9, November 3, 2016
cause the significant inhibition of neurosphere formation under
the same conditions (Figures S3E–S3G).
BrdU incorporation analysis showed that expression of NS4A,
NS4B, or NS4A-NS4B also altered the proliferation rates of
fNSCs (Figures 2D–2F) without affecting the expression of fNSCs
markers, including SOX2 (Figure 2A). Specifically, the expression
of NS4A, NS4B, or NS4A-NS4B led to a 48.9%, 44.1%, or 64.7%
reduction of fNSC proliferation, respectively, compared to the
vector control (Figure 2D). In addition, immunostaining for Nestin
and Ki-67 showed that expression of NS4A, NS4B, or NS4A-
NS4B led to a �43%, �28%, or �63% reduction of Nestin+-
and Ki-67+-positive proliferating fNSCs, respectively, compared
with that of vector control (Figures 2E and 2F). More interestingly,
when fNSCs were cultured on poly-L-ornithine- and laminin-
coated surface for 10 days to induce their differentiation into
neuronal cells (Guo et al., 2013; Wang et al., 2010), fNSCs
expressing NS4A, NS4B, or NS4A-NS4B poorly differentiated
into neurons or astrocytes (Figures 2G–2J). Upon expression of
NS4A, NS4B, or NS4A-NS4B, the differentiation rates to the
b3-tubulin-positive neuronal cells and GFAP-positive astrocytes
were reduced by approximately 25%–54% and 28%–51%,
respectively (Figures 2H and 2J). However, expression of
NS4A, NS4B, or NS4A-NS4B did not lead to apoptotic cell death
in fNSCs, suggesting that expression of NS4A and NS4B is not
toxic to cells (Figure S3H). Collectively, these data demonstrate
that mitotic neurogenesis of fNSCs is selectively and substan-
tially impaired by ZIKV NS4A and NS4B when these proteins
are ectopically expressed individually and in combination.
ZIKVNS4A andNS4B Induce Autophagy in Human fNSCsSince ZIKV infection induces autophagy inmultiple cell types, we
attempted to determine which viral proteins trigger that process.
Transient expression of individual ZIKV genes in HeLa-GFP-LC3
cells showed that the expression of either NS4A or NS4B had
marginal effects on GFP-LC3 punctate formation, whereas the
co-expression of both NS4A and NS4B led to a significant in-
crease in GFP-LC3 punctate numbers per cell (Figures 3A–3B
and S4A–S4D). Furthermore, when fNSCs or HeLa cells stably
expressing vector, NS4A, NS4B, or NS4A-NS4B were used,
expression of NS4A-NS4B also resulted in a significant increase
of LC3 I/II conversion in the presence or absence of lysosome
inhibitor bafilomycin A1 (Figures 3C–3F, S4E, and S4F). In addi-
tion, NS4A interacted with NS4B in cells (Figure S4H), and the
partial colocalizations between GFP-LC3 and NS4A or NS4B
were also detected (Figure S4G). These results show that ZIKV
NS4A and NS4B collaborate to induce efficient autophagy.
ZIKV NS4A and NS4B Inhibit Akt-mTOR SignalingAkt-mTOR signaling is essential for neurogenesis by human
fNSCs as well as for the induction of autophagy. Specifically,
Akt phosphorylation at Thr308 and Ser473 is required for its
full kinase activity (Chan et al., 2014) and subsequently, Akt-
mediated mTOR phosphorylation at Ser2448 is essential for
keeping autophagy in check. Our results show that ZIKV replica-
tion led to the suppression of Akt phosphorylation at both Thr308
and Ser473, which subsequently led to the reduction of mTOR
phosphorylation at Ser2448 (Figures 4A and S4I). Further
screening of individual ZIKV protein revealed that expression of
either NS4A or NS4B detectably reduced Akt phosphorylation
NS4A
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Figure 2. ZIKV NS4A and NS4B Impair Neurogenesis of fNSCs(A) Representative images of neurospheres formed at 7 dpi from fNSCs transduced with lentiviruses expressing ZIKV NS4A, NS4B, NS4A-NS4B, or vector alone.
Neurospheres were stained with fNSC markers Nestin and SOX2; upper panels represent primary neurospheres (P0), lower panels represent secondary neu-
rospheres after passage (P1). Scale bar, 50 mm.
(B) Quantification of number of neurospheres formed per 1 3 105 fNSCs transduced with lentiviruses as indicated. Mean ± SEM; p < 0.05 by one-way ANOVA.
(C) Percentage bar graph showing the distribution of neurospheres at different size ranges as described in (A).
(D) BrdU incorporation into fNSC-based flow cytometry analysis at 5 days after transductionwith different lentiviruses as indicated. Mean ± SEM; p < 0.05 by one-
way ANOVA.
(E and F) Representative confocal images of Nestin and Ki-67 double staining (E) and the quantification of proliferating Nestin+ and Ki-67+ double positive fNSCs
(F) at 5 days after transduction with different lentiviruses as indicated. Mean ± SEM; p < 0.05 by one-way ANOVA in (F).
(G and H) Representative confocal images of b3-tubulin immunostaining on fNSCs transduced with different lentiviruses as indicated followed by 10 days of
differentiation (G) and quantification (percentage) of b3-tubulin-positive neurons differentiated from fNSCs (H). Dapi: nucleus staining. Mean ± SEM; p < 0.05 by
one-way ANOVA in (H).
(I and J) Representative confocal images of GFAP immunostaining and nucleus staining with Dapi (I) and the quantification (percentage) of GFAP-positive as-
trocytes differentiated from fNSCs (J), 10 days after transduction with different lentiviruses as indicated. Mean ± SEM; p < 0.05 by one-way ANOVA in (J).
See also Figure S3.
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
at both Thr308 and Ser473 under normal conditions (Figure 4B).
When starved fNSCs or HeLa cells stably expressing vector,
NS4A, NS4B, or NS4A-NS4B were stimulated with serum or in-
sulin, expression of either NS4A or NS4B detectably suppressed
the Thr308 and Ser437 phosphorylations of Akt, and co-expres-
sion of NS4A and NS4B drastically suppressed the Thr308 and
Ser473 phosphorylations of Akt (Figures 4C–4E). Consequently,
co-expression of NS4A and NS4B resulted in the reduced levels
of mTOR phosphorylation at Ser2448 (Figures 4C–4E). To
confirm that the increased autophagy by NS4A-NS4B was
caused by impaired Akt/mTOR signaling, we expressed the
constitutively active form of Akt3 (myr-HA-Akt3 E17K) in fNSCs
to examine whether it blocked ZIKV infection- or NS4A-NS4B-
expression-mediated autophagy (Baek et al., 2015). As shown
in Figures S4J and S4K, ectopic expression of the constitutively
active form of Akt3 suppressed ZIKV infection- or NS4A-NS4B-
expression-mediated autophagy. These results suggest that
ZIKV NS4A and NS4B inhibit the Akt-mTOR signaling pathway,
Cell Stem Cell 19, 1–9, November 3, 2016 5
GF
P-L
C3 p
un
ctate/cell
IB: actin
IB: LC3
[Hela]
IB: actin
IB: LC3
[fNSC]
NS1 NS2A
NS2B NS3
Vector
NS4A
[H
ela-G
FP
-L
C3]
NS4B NS4A+NS4B
Capsid
NS1
NS5
E
Envelope M
A
C
B
D
0
5
10
15
20
E F
0
1
2
3
4
LC
3-II/L
C3-I
0
1
2
3
4
LC
3-II/L
C3-I
p<0.05
LC3-I
LC3-II
LC3-I
LC3-II
Figure 3. ZIKV NS4A and NS4B Induce Autophagy
(A and B) Screening of ZIKV proteins for autophagy induction. HeLa-GFP-LC3 cells transiently expressed each ZIKV protein as indicated by lentivirus infection.
The levels of GFP-LC3 puncta were measured and quantified at 2 dpi. Mean ± SEM; p < 0.05 by one-way ANOVA in (B).
(C–F) LC3 processing from fNSCs or HeLa cells stably expressing vector, NS4A, NS4B, or NS4A-NS4B was measured by immunoblot with indicated antibodies.
The levels of LC3-II/LC3-I were quantified by band intensity with Image Lab software (BioRad).
See also Figure S4.
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
which in turn impedes the neurogenesis of fNSCs and increases
autophagy.
DISCUSSION
Recent ZIKV outbreaks in South andCentral America and the un-
expected association between ZIKV infection and birth defects
have attracted global attention to the need to study the patho-
genesis of ZIKV-associated microcephaly and to develop thera-
peutic interventions against it. Recent reports demonstrated that
ZIKV infection impairs growth in iPSC-derived human neuro-
spheres and brain organoids (Garcez et al., 2016; Qian et al.,
2016). Infection of different mouse models has revealed that
ZIKV infection can cause neurological development anomalies
in mice as well (Lazear et al., 2016; Li et al., 2016; Miner et al.,
2016; Rossi et al., 2016). Yet to date, no detailed molecular
mechanisms underlying ZIKV pathogenesis have been unveiled,
hence hindering the development of ZIKV-targeted anti-viral
therapy. In this paper, we utilized NSCs isolated from human fe-
tuses between 18 and 22 weeks of gestational age to study how
ZIKV infection impairs growth and neurogenesis of fNSCs. By
screening the three structural proteins and seven NSs of ZIKV,
we found that the cooperation of NS4A and NS4B strongly sup-
presses host Akt-mTOR signaling, consequently leading to the
6 Cell Stem Cell 19, 1–9, November 3, 2016
impairment of neurogenesis of human fNSCs and the upregula-
tion of autophagy for viral replication.
Autophagy is a lysosome-mediated catabolic process that
also developed as an important ancient immune response during
evolution. Although hosts have evolved autophagy to maintain
cellular homeostasis and limited pathogen infection, some path-
ogens such as flaviviruses usurp cellular autophagy pathways to
benefit their life cycles. ZIKV infection induces autophagy in
multiple cell types including fNSCs. Our results demonstrate
that the autophagy inducer rapamycin increases ZIKV replica-
tion, whereas the autophagy inhibitor 3-MA or chloroquine de-
creases ZIKV replication. Although these inducers or inhibitors
also affect additional pathways besides autophagy, genetic KO
or knockdown of several autophagy genes shows the specific
suppression of ZIKV replication, suggesting that an efficient
replication of ZIKV depends on the autophagy pathway. In
fact, DENV replication requires autophagy to control processing
of lipid droplets and triglycerides (Heaton and Randall, 2010) and
HCV uses autophagosomal membranes as sites for its RNA
replication (Sir et al., 2012). Thus, these findings suggest that
ZIKV may also require host autophagy pathways to create the
membrane structures to serve as viral replication sites.
By screening the ten ZIKV-encoding potential proteins, we
found that NS4A and NS4B cooperate to induce efficient
actin
Akt
Akt pS473
Akt pT308
NS
4A
NS
4B
NS
4A
+N
S4B
vecto
r
NS
4A
NS
4B
NS
4A
+N
S4B
vecto
r
PBS Serum
[Hela]
mTOR
mTOR
pS2448
actin
Akt
Akt pS473
Akt pT308
NS
4A
NS
4B
NS
4A
+N
S4B
vecto
r
NS
4A
NS
4B
NS
4A
+N
S4B
vecto
r
PBS Insulin
[Hela]
mTOR
mTOR
pS2448
A
E B
D
[fNSC]
actin
Akt
Akt pS473
Akt pT308
NS
4A
NS
4B
NS
4A
+N
S4B
vecto
r
NS
4A
NS
4B
NS
4A
+N
S4B
vecto
r
PBS Insulin
mTOR
mTOR
pS2448
actin
Akt
Akt pS473
Akt pT308
mTOR
mTOR
pS2448
ZIKV 0 10 30 120 240 min
[fNSC]
C
actin
Akt
Akt pS473
Akt pT308
mTOR
mTOR
pS2448
NS
1
NS
2A
NS
2B
NS
3H
NS
3S
NS
4A
NS
4B
vecto
r
C
M
E
NS
3
NS
5
vecto
r
[Hela]
Figure 4. ZIKV NS4A and NS4B Inhibit the Akt-mTOR Signaling Pathway
(A) ZIKV replication inhibits Akt-mTOR signaling. fNSCs were infected with ZIKV strain MR766 at MOI 0.5. Cell lysates were harvested at various time points and
subjected to immunoblot with indicated antibodies.
(B) The levels of Akt andmTOR activities of HeLa cells expressing each ZIKV gene were measured with indicated antibodies. NS3H, NS3 helicase domain; NS3S,
NS3 serine protease domain.
(C–E) fNSCs or HeLa cells stably expressing vector, NS4A, NS4B, or NS4A-NS4B were stimulated with serum (20%) or insulin (2 mg/mL) after 8 hr starvation. Cell
lysates were harvested and subjected to immunoblot with indicated antibodies.
See also Figure S4.
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
autophagy by suppressing the Akt-mTOR signaling pathway
that is essential for controlling stimulation-induced autophagy.
Similar to DENV NS4A and NS4B, ZIKV NS4A and NS4B
are small hydrophobic proteins with potential transmembrane
spanning regions (Zou et al., 2015). While DENV NS4A alone in-
duces autophagy (McLean et al., 2011), an individual expres-
sion of ZIKV NS4A or NS4B has weak effects on autophagy,
suggesting that ZIKV utilizes different molecular mechanisms
from DENV to induce autophagy. While expression of the
constitutively active Akt fully suppressed either ZIKV infection-
or NS4A-NS4B-expression-mediated autophagy, it is possible
that the mechanism of ZIKV infection-mediated autophagy is
not entirely identical to that of NS4A-NS4B-expression-medi-
ated autophagy. Future studies with the reverse genetic anal-
ysis of ZIKV are needed to address how NS4A and NS4B
target and inhibit Akt in the context of viral genome (Shan
et al., 2016).
The Akt-mTOR signaling pathway is critical not only for con-
trolling autophagy induction, but also for cortical development
(Franke, 2008). Mutations in this pathway lead to several disor-
ders, such as megalencephaly by the constitutive activation of
Akt and microcephaly by the loss of function of Akt (Mirzaa
et al., 2013). Our data suggest that ZIKV NS4A and NS4B sup-
press the activation of Akt, consequently leading to the impair-
ment of proliferation and differentiation of fNSCs in vitro. These
may serve as a potential molecular mechanism by which ZIKV
infection can lead to neurological disorders such as micro-
cephaly. Due to its crucial role in cell growth and differentiation,
Akt activity is tightly controlled by post-translational modifica-
tions. Phosphorylations on Thr308 by PDK1 and on Ser473 by
mTORC2 are important for Akt activation, and phosphatases
such as PTEN, PP2A, and PHLPP antagonize Akt activation.
Other modifications such as ubiquitination, SUMOylation, acety-
lation, and O-GlcNAcylation have been also shown to positively
Cell Stem Cell 19, 1–9, November 3, 2016 7
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
or negatively regulate Akt activities (Chan et al., 2014). Our study
suggests that ZIKV NS4A and NS4B block Akt activation by in-
hibiting upstream PI3K signaling and potentially modulating
Akt post-translational modifications as well. Further studies will
be required to parse the specific means by which ZIKV NS4A
and NS4B influence Akt activation.
It should be noted that we utilized primary fNSCs recovered
from human fetuses to demonstrate how three ZIKV strains
(MR766, IbH30656, and H/PF/2013) can impair the growth and
neurogenesis of human fNSCs. Regardless of the viral strains
and fNSC isolates, similar levels of neurogenesis inhibition and
autophagy induction were observed. However, it is possible
that the ongoing epidemic ZIKV strains in Brazil and other South
American countries may cause more severe defects in the pro-
liferation of fNSCs and subsequent differentiation during corti-
cogenesis. On the other hand, since the sequences of NS4A
and NS4B are almost identical across various ZIKV strains,
additional factors might also be associated with pathogenesis
of the ongoing epidemic ZIKV strains. Additional studies are
ongoing to demonstrate the functions of NS4A and NS4B in
in vitro brain organoid models and in vivo IFNAR1 KO mouse
models. In summary, our research provides new mechanistic in-
sights on how NS4A and NS4B phenocopy ZIKV pathogenesis
in vitro, implicating them as potential anti-ZIKV therapeutic
targets.
EXPERIMENTAL PROCEDURES
Viruses, Plasmids, and Cell Culture
ZIKV strain MR766 (Uganda, 1947) and H/PF/2013 (French Polynesia, 2013)
were kindly provided by Dr. Michael Diamond (Washington University School
of Medicine) and Dr. Cecile Baronti (Aix Marseille Universite). ZIKV strain
IbH30656 was purchased from ATCC. ZIKV stocks were propagated in Vero
cells or C6/36 Aedes albopictus cells after being inoculated at an MOI of
0.02 and supernatants were harvested at 96 hpi. The titers of ZIKV stocks
were determined by plaque assay on Vero cells as described previously (Liang
et al., 2014).
ZIKV cDNA was synthesized as DNA fragments (IDT) and ZIKV expression
constructs were amplified by PCR and cloned into lentiviral pCDH-puro
or pCDH-Hyg vectors with the N-terminal Flag tag. All constructs were
sequenced using an ABI PRISM 377 automatic DNA sequencer to verify
100% correspondence with the original sequence.
HeLa cells, HEK293T cells, and MEFs were maintained in DMEM (GIBCO-
BRL) containing 4 mM glutamine and 10% FBS. Vero cells were cultured in
DMEM with 5% FBS and 4 mM glutamine. C6/36 Aedes albopictus cells
were cultured in DMEMwith 10%FBS, non-essential amino acids, and HEPES
at 28� with 5% CO2. Transient transfections were performed with Lipofect-
amine 2000 (Invitrogen). HeLa stable cell lines were generated using a stan-
dard selection protocol with puromycin (2 mg/mL) for individual expressions
of ZIKV gene, or puromycin with hygromycin (200 mg/mL) for the co-expression
of NS4A-NS4B.
SUPPLEMENTAL INFORMATION
Supplemental Information for this article includes four figures and Supple-
mental Experimental Procedures and can be found with this article online at
http://dx.doi.org/10.1016/j.stem.2016.07.019.
AUTHOR CONTRIBUTIONS
Q.L., Z.Z., and J.U.J conceived of the research, designed the study, and wrote
the manuscript. Q.L., Z.Z., Z.L., J.Z., W.C., S.-S.F., S.-A.L., and J.G. per-
formed experiments and analyzed data. S.W., S.A.G., and B.V.Z. provided
human fNSCs. All authors commented on the manuscript.
8 Cell Stem Cell 19, 1–9, November 3, 2016
ACKNOWLEDGMENTS
We specially thank Michael Diamond and Cecile Baronti for providing ZIKV
strains MR766 and H/PF/2013. This work was partly supported by
CA200422, CA180779, DE023926, AI073099, AI116585, HL110609, Hastings
Foundation, Fletcher Jones Foundation, and GRL Program (K20815000001)
from National Research Foundation of Korea (JUJ); NIRG-15-363387 from
the Alzheimer’s Association (Z.Z.); 9R01NS090904 (B.V.Z.); and the Cure for
Alzheimer’s Fund (B.V.Z. and Z.Z.). Q.L. is a Special Fellow of Leukemia &
Lymphoma Society and is supported by the Program for Professor of Special
Appointment (Eastern Scholar) at Shanghai Institutions of Higher learning.
Received: May 10, 2016
Revised: June 20, 2016
Accepted: July 21, 2016
Published: August 11, 2016
REFERENCES
Baek, S.T., Copeland, B., Yun, E.-J., Kwon, S.-K., Guemez-Gamboa, A.,
Schaffer, A.E., Kim, S., Kang, H.-C., Song, S., Mathern, G.W., and Gleeson,
J.G. (2015). An AKT3-FOXG1-reelin network underlies defective migration in
human focal malformations of cortical development. Nat. Med. 21, 1445–1454.
Buchkovich, N.J., Yu, Y., Zampieri, C.A., and Alwine, J.C. (2008). The TORrid
affairs of viruses: effects of mammalian DNA viruses on the PI3K-Akt-mTOR
signalling pathway. Nat. Rev. Microbiol. 6, 266–275.
Calvet, G., Aguiar, R.S., Melo, A.S.O., Sampaio, S.A., de Filippis, I., Fabri, A.,
Araujo, E.S.M., de Sequeira, P.C., de Mendonca, M.C.L., de Oliveira, L., et al.
(2016). Detection and sequencing of Zika virus from amniotic fluid of fetuses
with microcephaly in Brazil: a case study. Lancet Infect. Dis. 16, 653–660.
Cauchemez, S., Besnard, M., Bompard, P., Dub, T., Guillemette-Artur, P.,
Eyrolle-Guignot, D., Salje, H., Van Kerkhove, M.D., Abadie, V., Garel, C.,
et al. (2016). Association between Zika virus and microcephaly in French
Polynesia, 2013-15: a retrospective study. Lancet 387, 2125–2132.
Chan, C.-H., Jo, U., Kohrman, A., Rezaeian, A.H., Chou, P.-C., Logothetis, C.,
and Lin, H.-K. (2014). Posttranslational regulation of Akt in human cancer. Cell
Biosci. 4, 59.
Cloetta, D., Thomanetz, V., Baranek, C., Lustenberger, R.M., Lin, S., Oliveri, F.,
Atanasoski, S., and Ruegg, M.A. (2013). Inactivation of mTORC1 in the devel-
oping brain causes microcephaly and affects gliogenesis. J. Neurosci. 33,
7799–7810.
Cugola, F.R., Fernandes, I.R., Russo, F.B., Freitas, B.C., Dias, J.L., Guimaraes,
K.P., Benazzato, C., Almeida, N., Pignatari, G.C., Romero, S., et al. (2016). The
Brazilian Zika virus strain causes birth defects in experimental models. Nature
534, 267–271.
DeCarvalho, N.S., DeCarvalho, B.F., Fugaca, C.A., Doris, B., andBiscaia, E.S.
(2016). Zika virus infection during pregnancy and microcephaly occurrence: a
review of literature and Brazilian data. Braz. J. Infect. Dis. 20, 282–289.
Dick, G.W.A. (1952). Zika virus. II. Pathogenicity and physical properties.
Trans. R. Soc. Trop. Med. Hyg. 46, 521–534.
Dick, G.W.A., Kitchen, S.F., and Haddow, A.J. (1952). Zika virus. I. Isolations
and serological specificity. Trans. R. Soc. Trop. Med. Hyg. 46, 509–520.
Duffy, M.R., Chen, T.-H., Hancock, W.T., Powers, A.M., Kool, J.L., Lanciotti,
R.S., Pretrick, M., Marfel, M., Holzbauer, S., Dubray, C., et al. (2009). Zika virus
outbreak on Yap Island, Federated States of Micronesia. N. Engl. J. Med. 360,
2536–2543.
Franke, T.F. (2008). PI3K/Akt: getting it right matters. Oncogene 27, 6473–
6488.
Garcez, P.P., Loiola, E.C., Madeiro da Costa, R., Higa, L.M., Trindade, P.,
Delvecchio, R., Nascimento, J.M., Brindeiro, R., Tanuri, A., and Rehen, S.K.
(2016). Zika virus impairs growth in human neurospheres and brain organoids.
Science 352, 816–818.
Gotz, M., and Huttner, W.B. (2005). The cell biology of neurogenesis. Nat. Rev.
Mol. Cell Biol. 6, 777–788.
Please cite this article in press as: Liang et al., Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cellsto Inhibit Neurogenesis and Induce Autophagy, Cell Stem Cell (2016), http://dx.doi.org/10.1016/j.stem.2016.07.019
Guo, H., Zhao, Z., Yang, Q., Wang, M., Bell, R.D., Wang, S., Chow, N., Davis,
T.P., Griffin, J.H., Goldman, S.A., and Zlokovic, B.V. (2013). An activated pro-
tein C analog stimulates neuronal production by human neural progenitor cells
via a PAR1-PAR3-S1PR1-Akt pathway. J. Neurosci. 33, 6181–6190.
Hamel, R., Dejarnac, O.,Wichit, S., Ekchariyawat, P., Neyret, A., Luplertlop, N.,
Perera-Lecoin, M., Surasombatpattana, P., Talignani, L., Thomas, F., et al.
(2015). Biology of Zika Virus Infection in Human Skin Cells. J. Virol. 89,
8880–8896.
Heaton, N.S., and Randall, G. (2010). Dengue virus-induced autophagy regu-
lates lipid metabolism. Cell Host Microbe 8, 422–432.
Heymann, D.L., Hodgson, A., Sall, A.A., Freedman, D.O., Staples, J.E.,
Althabe, F., Baruah, K., Mahmud, G., Kandun, N., Vasconcelos, P.F.C., et al.
(2016). Zika virus and microcephaly: why is this situation a PHEIC? Lancet
387, 719–721.
Keyoung, H.M., Roy, N.S., Benraiss, A., Louissaint, A., Jr., Suzuki, A.,
Hashimoto, M., Rashbaum, W.K., Okano, H., and Goldman, S.A. (2001).
High-yield selection and extraction of two promoter-defined phenotypes of
neural stem cells from the fetal human brain. Nat. Biotechnol. 19, 843–850.
Lazear, H.M., Govero, J., Smith, A.M., Platt, D.J., Fernandez, E., Miner, J.J.,
and Diamond, M.S. (2016). A Mouse Model of Zika Virus Pathogenesis. Cell
Host Microbe 19, 720–730.
Lee, Y. (2015). Roles of mTORSignaling in Brain Development. Exp. Neurobiol.
24, 177–185.
Levine, B., Mizushima, N., and Virgin, H.W. (2011). Autophagy in immunity and
inflammation. Nature 469, 323–335.
Li, C., Xu, D., Ye, Q., Hong, S., Jiang, Y., Liu, X., Zhang, N., Shi, L., Qin, C.-F.,
and Xu, Z. (2016). Zika virus disrupts neural progenitor development and leads
to microcephaly in mice. Cell Stem Cell 19, 120–126.
Liang, Q., Chang, B., Brulois, K.F., Castro, K., Min, C.-K., Rodgers, M.A., Shi,
M., Ge, J., Feng, P., Oh, B.-H., and Jung, J.U. (2013). Kaposi’s sarcoma-asso-
ciated herpesvirus K7 modulates Rubicon-mediated inhibition of autophago-
some maturation. J. Virol. 87, 12499–12503.
Liang, Q., Seo, G.J., Choi, Y.J., Kwak, M.-J., Ge, J., Rodgers, M.A., Shi, M.,
Leslie, B.J., Hopfner, K.-P., Ha, T., et al. (2014). Crosstalk between the
cGASDNA sensor andBeclin-1 autophagy protein shapes innate antimicrobial
immune responses. Cell Host Microbe 15, 228–238.
Liang, Q., Chang, B., Lee, P., Brulois, K.F., Ge, J., Shi, M., Rodgers, M.A.,
Feng, P., Oh, B.-H., Liang, C., and Jung, J.U. (2015). Identification of the
Essential Role of Viral Bcl-2 for Kaposi’s Sarcoma-Associated Herpesvirus
Lytic Replication. J. Virol. 89, 5308–5317.
McLean, J.E., Wudzinska, A., Datan, E., Quaglino, D., and Zakeri, Z. (2011).
Flavivirus NS4A-induced autophagy protects cells against death and en-
hances virus replication. J. Biol. Chem. 286, 22147–22159.
Miner, J.J., Cao, B., Govero, J., Smith, A.M., Fernandez, E., Cabrera, O.H.,
Garber, C., Noll, M., Klein, R.S., Noguchi, K.K., et al. (2016). Zika Virus
Infection during Pregnancy in Mice Causes Placental Damage and Fetal
Demise. Cell 165, 1081–1091.
Ming, G.L., and Song, H. (2011). Adult neurogenesis in the mammalian brain:
significant answers and significant questions. Neuron 70, 687–702.
Mirzaa, G.M., Riviere, J.-B., and Dobyns, W.B. (2013). Megalencephaly syn-
dromes and activating mutations in the PI3K-AKT pathway: MPPH and
MCAP. Am. J. Med. Genet. C. Semin. Med. Genet. 163C, 122–130.
Mlakar, J., Korva, M., Tul, N., Popovi�c, M., Polj�sak-Prijatelj, M., Mraz, J.,
Kolenc, M., Resman Rus, K., Vesnaver Vipotnik, T., Fabjan Vodu�sek, V.,
et al. (2016). Zika Virus Associated with Microcephaly. N. Engl. J. Med. 374,
951–958.
Musso, D., Roche, C., Robin, E., Nhan, T., Teissier, A., and Cao-Lormeau,
V.-M. (2015). Potential sexual transmission of Zika virus. Emerg. Infect. Dis.
21, 359–361.
Nellist, M., Schot, R., Hoogeveen-Westerveld, M., Neuteboom, R.F., van der
Louw, E.J.T.M., Lequin, M.H., Bindels-de Heus, K., Sibbles, B.J., de Coo,
R., Brooks, A., and Mancini, G.M. (2015). Germline activating AKT3 mutation
associated with megalencephaly, polymicrogyria, epilepsy and hypoglycemia.
Mol. Genet. Metab. 114, 467–473.
Qian, X., Nguyen, H.N., Song, M.M., Hadiono, C., Ogden, S.C., Hammack, C.,
Yao, B., Hamersky, G.R., Jacob, F., Zhong, C., et al. (2016). Brain-Region-
Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell
165, 1238–1254.
Riviere, J.-B., Mirzaa, G.M., O’Roak, B.J., Beddaoui, M., Alcantara, D.,
Conway, R.L., St-Onge, J., Schwartzentruber, J.A., Gripp, K.W., Nikkel,
S.M., et al.; Finding of Rare Disease Genes (FORGE) Canada Consortium
(2012). De novo germline and postzygotic mutations in AKT3, PIK3R2
and PIK3CA cause a spectrum of related megalencephaly syndromes. Nat.
Genet. 44, 934–940.
Rossi, S.L., Tesh, R.B., Azar, S.R., Muruato, A.E., Hanley, K.A., Auguste, A.J.,
Langsjoen, R.M., Paessler, S., Vasilakis, N., and Weaver, S.C. (2016).
Characterization of a Novel Murine Model to Study Zika Virus. Am. J. Trop.
Med. Hyg. 94, 1362–1369.
Shan, C., Xie, X., Muruato, A.E., Rossi, S.L., Roundy, C.M., Azar, S.R., Yang,
Y., Tesh, R.B., Bourne, N., Barrett, A.D., et al. (2016). An Infectious cDNA
Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and
Antiviral Inhibitors. Cell Host Microbe 19, 891–900.
Simpson, D.I.H. (1964). Zika virus infection in man. Trans. R. Soc. Trop. Med.
Hyg. 58, 335–338.
Sir, D., Kuo, C.F., Tian, Y., Liu, H.M., Huang, E.J., Jung, J.U., Machida, K., and
Ou, J.-H.J. (2012). Replication of hepatitis C virus RNA on autophagosomal
membranes. J. Biol. Chem. 287, 18036–18043.
Tang, H., Hammack, C., Ogden, S.C., Wen, Z., Qian, X., Li, Y., Yao, B., Shin, J.,
Zhang, F., Lee, E.M., et al. (2016). Zika Virus Infects Human Cortical Neural
Progenitors and Attenuates Their Growth. Cell Stem Cell 18, 587–590.
Venturi, G., Zammarchi, L., Fortuna, C., Remoli, M.E., Benedetti, E., Fiorentini,
C., Trotta, M., Rizzo, C., Mantella, A., Rezza, G., and Bartoloni, A. (2016). An
autochthonous case of Zika due to possible sexual transmission, Florence,
Italy, 2014. Euro Surveill. 21, 21.
Wahane, S.D., Hellbach, N., Prentzell, M.T., Weise, S.C., Vezzali, R., Kreutz,
C., Timmer, J., Krieglstein, K., Thedieck, K., and Vogel, T. (2014). PI3K-
p110-alpha-subtype signalling mediates survival, proliferation and neurogen-
esis of cortical progenitor cells via activation of mTORC2. J. Neurochem. 130,
255–267.
Wang, S., Chandler-Militello, D., Lu, G., Roy, N.S., Zielke, A., Auvergne, R.,
Stanwood, N., Geschwind, D., Coppola, G., Nicolis, S.K., et al. (2010).
Prospective identification, isolation, and profiling of a telomerase-expressing
subpopulation of human neural stem cells, using sox2 enhancer-directed fluo-
rescence-activated cell sorting. J. Neurosci. 30, 14635–14648.
Williams, L.R., and Taylor, G.S. (2012). Autophagy and immunity - insights from
human herpesviruses. Front. Immunol. 3, 170.
Zou, J., Xie, X., Wang, Q.-Y., Dong, H., Lee, M.Y., Kang, C., Yuan, Z., and Shi,
P.-Y. (2015). Characterization of dengue virus NS4A and NS4B protein interac-
tion. J. Virol. 89, 3455–3470.
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