Zika e proteinas

Short Article

Zika Virus NS4A and NS4B
Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cells toInhibit Neurogenesis and Induce Autophagy
Graphical 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.

mailto:[email protected]



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

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.)


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





Mock MR766

x y

z

x y

z

A B

C

D

F G

x y

z

Nes

tinS

ox2T

UN

EL

x y

z

0

20

40

60

% C

ell d

eath

(5 d

pi)

Mock

MR766

p<0.05

IbH30

656

0

30

60

90

Mock

p<0.01

Neu

rosp

here

siz

e(d

iam

eter

at 3

dpi

, μm

)

MR766

IbH30

656

Mock MR766

IbH30656

Mock MR766

0

500

750

1000

Mock

p<0.05

MR766

IbH30

656N

umbe

r of n

euro

sphe

res

for

med

/ 10

ce

lls5

250

EH/P

F/2013Li

ve /

Dea

d A

ssay

Mock MR766 IbH30656 H/PF/2013

H/PF/2013

H/PF/20

13

H/PF/20

13

IB: actin

IB: LC3

IB: p62

0 6 12 24 0 12 24 h

[fNSC]

LC3-I LC3-II

MR766 IbH30656

0

20

40

60

80

% c

ells

with

LC

3 pu

ncta

0

5

10

15

20

25 p < 0.05

LC3

punc

ta /

cell

LC3 LC3 Nestin

Moc

kM

R76

6Ib

H30

656

p < 0.05

Mock

MR766

IbH30

656

H

J

I

Rel

ativ

e ZI

KV

RN

A le

vel

0

10

20

30

40

50WaterRapamycinChloroquine3-MA

Mock MR766 IbH30656

Nes

tin/S

ox2/

ZIK

V

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



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


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


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

NS4B NS4A+4B

Vector

GFA

P D

api

GFA

P D

api

p<0.05

0

20

40

60

80

NS4ANS4B

NS4A+4

BVec

tor

% G

FAP

Dap

i ce

lls+

+NS4A

NS4B

NS4A+4

BVec

tor

% β

3-tu

bulin

Dap

i ce

lls+

+

0

20

40

60 p<0.05NS4AVector

β3-tu

bulin

Dap

iβ3

-tubu

lin D

api NS4B NS4A+4B

D

G H I J

E

0

5

10

15

%B

rdU

Inco

rpor

atio

n

NS4ANS4B

NS4A+4

BVec

tor

p<0.01

NS4AP0

P1

NS4B NS4A+4BVector

Nes

tinS

ox2

Nes

tinS

ox2

A B

0

25%

50%

75%

100%P0 P1

>150100-15050-100<50

NS4ANS4B

NS4A+4

BVec

torNS4A

NS4B

NS4A+4

BVec

torNeu

rosp

here

siz

e di

strib

utio

n

0

20

40

60

80

NS4ANS4B

NS4A+4

BVec

tor

% K

i67

Nes

tin c

ells

++

p<0.05F

Nes

tinK

i-67

Ki-6

7D

api

NS4A NS4B NS4A+4BVector

Num

ber o

f neu

rosp

here

s

f

orm

ed /

10

cells

5

NS4ANS4B

NS4A+4

BVec

tor

p<0.05

0

500

750

1000

250

C

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.


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,


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.


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


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.


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



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.


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.


http://refhub.elsevier.com/S1934-5909(16)30214-4/sref1














































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.


















































































































Zika e proteinas

Self Improvement

Transcript of Zika e proteinas