Abstract

   Zika virus (ZIKV) infection is an emerging global threat that is
   suspected to be associated with fetal microcephaly. However, the
   molecular mechanisms underlying ZIKV disease pathogenesis in humans
   remain elusive. Here, we investigated the human protein interaction
   network associated with ZIKV infection using a systemic virology
   approach, and reconstructed the transcriptional regulatory network to
   analyze the mechanisms underlying ZIKV-elicited microcephaly
   pathogenesis. The bioinformatics findings in this study show that P53
   is the hub of the genetic regulatory network for ZIKV-related and
   microcephaly-associated proteins. Importantly, these results imply that
   the ZIKV capsid protein interacts with mouse double-minute-2 homolog
   (MDM2), which is involved in the P53-mediated apoptosis pathway,
   activating the death of infected neural cells. We also found that
   synthetic mimics of the ZIKV capsid protein induced cell death in vitro
   and in vivo. This study provides important insight into the
   relationship between ZIKV infection and brain diseases.

   Keywords: Zika virus, microcephaly, capsid protein, P53, MDM2, cell
   death

Introduction

   Zika virus (ZIKV) is a single-stranded, positive-sense RNA virus
   belonging to the genus Flavivirus in the family Flaviviridae, which
   also includes Dengue virus, West Nile virus, Japanese encephalitis
   virus, and Yellow fever virus (Musso and Gubler, [47]2016; Weaver et
   al., [48]2016). Zika virus diseases (ZVD) are caused by ZIKV, an
   emerging mosquito-borne virus, and have threatened to become a global
   pandemic since 2015 (Fauci and Morens, [49]2016; Petersen et al.,
   [50]2016). Epidemiological evidence suggests that ZIKV infection in
   pregnant women in Brazil is associated with the increasing numbers of
   congenital microcephaly cases reported in that country (Calvet et al.,
   [51]2016; Cauchemez et al., [52]2016; Gabriel et al., [53]2017).
   However, a direct causal link between ZIKV and microcephaly has yet to
   be demonstrated (Cugola et al., [54]2016; Li et al., [55]2016; Miner et
   al., [56]2016). There is currently no vaccine or drug to prevent or
   treat ZIKV infection (Mlakar et al., [57]2016; Rasmussen et al.,
   [58]2016).

   Targeting the molecular factors in human cells that are related to ZVD
   is a potentially useful strategy for the development of therapeutic
   drugs for these conditions (Hamel et al., [59]2015). Biomarkers are
   also urgently required to verify the relationship between ZIKV
   infection and microcephaly (Adibi et al., [60]2016; Gabriel et al.,
   [61]2016). In this study, we investigated the molecular mechanisms that
   may be responsible for ZIKV-induced cell death during abnormal brain
   development. The genome sequences of ZIKV from the 2015 outbreak in
   humans provide us with a novel opportunity to predict the host factors
   that are required for infection by ZIKV (Ioos et al., [62]2014; Calvet
   et al., [63]2016; Cunha et al., [64]2016; Faria et al., [65]2016;
   Mlakar et al., [66]2016; Wang et al., [67]2016). It has been reported
   that the flaviviruses share a similar genomic organization and
   replication strategy, even though they cause a wide range of distinct
   clinical diseases in humans (Fernandez-Garcia et al., [68]2009). The
   viral proteins in the genus Flavivirus often interact with the same
   host proteins, so known flavivirus–host interactions may be useful in
   predicting ZIKV–human protein interactions (Huang et al., [69]2014;
   Wang et al., [70]2016). Furthermore, the protein similarities among the
   ZIKV-related viruses suggest that they interact with the same molecular
   factors in the host genome as does ZIKV. With the development of
   protein–protein interaction databases, computational approaches provide
   a viable alternative method of mapping genetic regulatory networks in
   the human genome (Pastorino et al., [71]2010; Shah et al., [72]2015).

   In this study, a database of 248 ZIKV-related proteins in the human
   genome and 221 microcephaly-associated human proteins was created, and
   used to predict the molecular mechanisms involved in ZIKV infection and
   microcephaly. The results of this study indicate that these related
   proteins manipulate the cell death pathway through specific protein
   interactions during the genetic evolution of ZIKV, leading to a
   developmental disorder of the neurological system. Mechanisms by which
   ZIKV may be associated with microcephaly are presented here to provide
   a set of hypotheses for the further experimental investigation of the
   molecular mechanisms underlying ZIKV-induced cell death and to suggest
   targets for potential therapeutic drugs.

Results

P53 is the hub of the genetic regulatory network for ZIKV-related and
microcephaly-associated proteins

   The reconstructed transcriptional regulatory network based on the
   predicted ZIKV-related and microcephaly-associated human proteins
   (Figures [73]S1–[74]S3) is shown in Figures [75]1A,B. The predicted
   upstream regulator of the ZIKV–human interaction proteins is related to
   brain development and brain diseases, such as neuronal cell death, the
   differentiation of neurons, the differentiation of the nervous system,
   and the development of the head (Figure [76]1A). The upstream
   regulators involved in viral infection clearly implicate the
   P53-dependent apoptotic pathway (Figure [77]1A), which interacts
   strongly with the transcriptional regulator in human cells to induce
   cell death in the brain. The reconstructed molecular regulatory network
   of the microcephaly-associated human proteins shows that the upstream
   regulatory factor is directly related to viral infection and
   microcephaly, which can damage the normal molecular regulation of the
   morphology of the head and brain, the development of head, and the
   differentiation of neurons, inducing an abnormal morphology of head
   (Figure [78]1B). Interestingly, the hub of this gene regulatory network
   is also the P53 protein (Figure [79]1B), which is the key co-regulator
   with P63, CCND1, CDK4, HIF1A, MDM2, and JUN in the P53 signaling
   pathway (Figures [80]1A,B).

Figure 1.

   [81]Figure 1
   [82]Open in a new tab

   Transcriptional regulatory network and P53 signaling pathways
   associated with ZIKV infection and microcephaly. (A,B) Transcriptional
   regulatory networks of ZIKV-related (A) and microcephaly-associated (B)
   human proteins.

P53 cell death pathways play important roles in ZIKV infection and
microcephaly

   To identify the possible mechanistic functional pathway connecting the
   ZIKV-related and microcephaly-associated human proteins,
   pathway-related information was inferred with a phenotype annotation
   tool and used to perform functional and disease pathway enrichment
   analyses (Figures [83]S4, [84]S5). The diverse pathways relevant to ZVD
   include the mechanisms involved in the viral exit from the host cells,
   viral entry via endocytic pathways, and the activation of NF-κB by
   viruses. The death receptor signaling, apoptosis signaling, p53
   signaling, and autophagy pathways are also implicated in the mechanisms
   of ZVD (Figure [85]S4A). The microcephaly-associated human proteins are
   functionally closely related and are involved in multiple pathways,
   which are shared by the proteins involved in the ZIKV–human
   interaction. These pathways involve P53 signaling, NF-κB activation by
   viruses, the mechanisms of viral exit from the host cells, apoptosis
   signaling, and death receptor signaling (Figure [86]S4B). These shared
   pathways are important in the human immune response to viral infection
   and cell apoptosis, especially the P53 signaling pathway. Notably, the
   finding that the ZIKV–human interaction proteins are involved in the
   death receptor signaling pathway implies that ZIKV infection induces
   brain disorders during fetal development via apoptosis or cell death,
   which are related to P53 signaling and the direct interaction of P53
   with the MDM2 protein (Yang et al., [87]2008; Bhuvanakantham et al.,
   [88]2010) in Figure [89]2. Figure [90]2 also provides detailed
   information about the key regulatory factors in the P53 signaling
   pathway that play important roles in the activation of apoptosis by
   viruses (Austin et al., [91]2012; Ghouzzi et al., [92]2016). These
   results suggest that ZIKV affects the MDM2-mediated P53 signaling
   pathway to induce programmed cell death, leading to defects in brain
   and neuron development.

Figure 2.

   [93]Figure 2
   [94]Open in a new tab

   Detailed information on the key regulatory factors involved in P53
   signaling that play important roles in the activation of apoptosis by
   viruses.

Interaction between ZCP and MDM2 is involved in P53-mediated apoptosis
pathways

   To determine the potential P53-induced cell death pathways regulated by
   MDM2 together with ZCP during ZVD, we used the strategy illustrated
   schematically in Figure [95]3A. The interaction region was identified
   as residues 50–65 (Figure [96]3B, purple helix; Figures [97]S7A–C in
   Supplementary Materials) in the MDM2 structure (PDB ID: 3W69), in
   molecular docking and previous publications (Miyazaki et al.,
   [98]2013). It is also the critical binding domain in the P53–MDM2
   interaction and acts as a ubiquitin protein ligase, suppressing the
   transcriptional activity of P53 and promoting its degradation. This
   finding, based on previous bioinformatics data, suggests that the
   C-terminal helix of the ZCP (residues 74–97; Figure [99]3C, purple
   helix) mediates the effects of P53-induced cell death on brain
   development through a similar interaction with MDM2 (Yang et al.,
   [100]2008; Bhuvanakantham et al., [101]2010).

Figure 3.

   [102]Figure 3
   [103]Open in a new tab

   Schematic diagram and structure of the interaction between the ZIKV
   capsid protein (ZCP) and MDM2. (A) Schematic diagram of the interaction
   between ZCP and MDM2 in the P53-mediated apoptosis pathways. (B)
   Binding domain (residues 50–65) in the MDM2 structure (PDB ID: 3W69)
   that is critical for the P53–MDM2 interaction is shown as a purple
   helix. (C) Homology modeling of ZCP, in which the C-terminal helix is
   colored purple (residues 74–97). (D) Strong hydrogen bonding is shown
   at the O of MET50 (ball-and-stick model) in MDM2, which binds to HZ2 of
   LYS85 (ball-and-stick model) in ZCP. Right purple helix is the mimic
   ZCP peptide (residues 74–97). (E) Match between residues 81–97 (ILE81,
   LYS82, PHE84, LYS85, LEU88, ALA89, ALA91, LEU92, ALA95, ASN96, and
   ALA97) in the C-terminal regions of ZCP and MDM2. MDM2 protein is shown
   in the protein surface style and the residues of the mimic peptide are
   shown with sticks.

   Our molecular docking results indicate that the C-termini of ZCP and
   MDM2 form a strong hydrogen bond, in which the oxygen (O) of MET50
   (ball-and-stick model) in MDM2 binds to the HZ2 of LYS85
   (ball-and-stick model) in the C-terminal region of ZCP (Figure [104]3D
   and Figure [105]S6). We also detected a hydrophobic interaction and
   steric matching between the C-termini of ZCP and MDM2. Residues 81–97
   (ILE81, LYS82, PHE84, LYS85, LEU88, ALA89, ALA91, LEU92, ALA95, ASN96,
   and ALA97) at the C-terminus of ZCP matched the MDM2 protein tightly
   (Figure [106]3E). These hydrophobic interactions contribute to the
   interaction between the two proteins and may also play a key role in
   P53-mediated cell death during brain development.

   To examine the interaction between ZCP and MDM2, we synthesized a
   peptide with the C-terminal sequence of ZCP (mimic peptide; residues
   74–97) to mimic ZCP. We used an enzyme-linked immunosorbent assay
   (ELISA) of human MDM2 to determine the concentration of free MDM2 in
   U87 cells after treatment with the synthetic peptide (Figure [107]4A).
   The results shown in Figure [108]4A indicate that the synthetic ZCP
   peptide reduced the cellular free MDM2 protein in the U87 cells,
   implying a probable direct interaction between ZCP and MDM2 in vivo. We
   then measured the activation of P53 and the cell death marker in U87
   cells after treatment with the ZCP mimic (Figures [109]4B–D). This
   induced the activation of P53 and the apoptosis marker in the protein
   array (Figures [110]4B,C). We detected significantly increased levels
   of cleaved caspase 3 and the phosphorylation of Ser15, Ser46, and
   Ser392 in P53, which are well-established markers of P53 activation.
   The result of a western blotting analysis also showed increases in
   total P53, cleaved caspase 9, and cleaved caspase 3 proteins, which
   confirmed the activation of the P53-dependent apoptosis pathway (Figure
   [111]4D). We selected a non-specific peptide derived from ZCP (residues
   30–53; Figure [112]S7D) as the control peptide in subsequent
   experiments, because it does not interact with MDM2. The non-specific
   peptide did not affect the levels of those proteins in the treated
   cells. Consistent with our previous predictive results and recent
   reports, these results suggest that the synthetic peptide functionally
   mimics ZCP in the MDM2-mediated activation of P53 during the cell death
   process.

Figure 4.

   [113]Figure 4
   [114]Open in a new tab

   Detection of the interaction between MDM2 and the synthetic ZCP peptide
   during the activation of P53-induced cell death in U87 cells. (A)
   Competitive inhibition effect of treatment with the synthetic peptide
   on intracellular free MDM2 in U87 cells. The concentration of cellular
   free MDM2 was determined with an ELISA. Values shown are means ± SD.
   (B) P53 activation and apoptotic markers were upregulated in the
   synthetic-peptide-treated U87 cells compared with the control in the
   protein array. (C) Quantification of the markers in the images shown in
   (B), signals were quantified with ImageJ. (D) Activation of total P53
   and apoptotic markers were confirmed with western blotting. (E)
   Non-specific peptide. SD, standard deviation. ^*P < 0.05; ^**P < 0.01.

Reduced cell viability and induction of apoptosis by mimic ZCP peptide in
culture

   To explore the potential involvement of ZCP in cell death in vitro, we
   first measured the viability of cultured U87 cells treated with the
   synthetic ZCP peptide, using a CCK-8 assay. The viability of the U87
   cells decreased significantly and dose-dependently after treatment with
   the mimic peptide (Figure [115]5A). The U87 cells were then incubated
   with different concentrations (5 or 10 μM) of the synthetic peptide to
   mimic ZCP-induced cell death in vitro. Six hours after the addition of
   the synthetic ZCP peptide to the serum-free medium, a microscopic
   analysis revealed morphological changes typical of apoptosis in all the
   mimic-peptide-treated cells, but not in the
   non-specific-peptide-treated cells (Figure [116]5B). Significant
   necrosis and apoptotic bodies were also observed in the
   mimic-peptide-treated cells (Figure [117]5B). As shown in Figure
   [118]5C, all the mimic-peptide-treated U87 cells stained with TUNEL,
   and almost all of them stained positively in their nuclei. Apoptotic
   U87 cells displaying fluorescent signals were clearly evident after
   treatment for 6 h, when the cells were viewed with fluorescence
   microscopy. However, TUNEL staining remained at the baseline level in
   the cells treated with the non-specific peptide. This finding indicates
   that the mimic-peptide-treated cells were apoptotic.

Figure 5.

   Figure 5
   [119]Open in a new tab

   U87 cells were treated with a synthetic peptide derived from ZCP. (A)
   Effect of the synthetic peptide on the viability of U87 cells is shown.
   U87 cells were exposed to different concentrations (0, 5, or 10 μM) of
   the synthetic peptide for 4 h and cell viability was determined with
   the CCK-8 assay. Representative result is shown as the mean ± SD of
   triplicate wells from one of three independent experiments. (B)
   Morphology of U87 cells after incubation for 0, 6, or 12 h at 37°C with
   various peptide concentrations (0, 5, or 10 μM). (C) Fluorescent
   signals for TUNEL staining (green) and DAPI (blue) in U87 cells treated
   with 0, 5, or 10 μM ZCP peptide for 4 h at 37°C. Bars: 100 μm. SD,
   standard deviation. ^*P < 0.05; ^**P < 0.001.

Synthetic ZCP mimic induces brain cell death in vivo

   To investigate whether ZCP induces brain injury in vivo, we injected
   the synthetic ZCP peptide directly into Balb/C mouse brains
   ([120]Supplemental Videos). At 6 h after injection, the brains from the
   Group B and C mice were hypertrophic and displayed only mild swelling
   compared with those of Group A. In a histopathological analysis,
   hematoxylin and eosin (HE)-stained brain tissues revealed a small
   amount of degenerating or necrotic neuronal cells and the occasional
   infiltration of a few inflammatory cells in the caudate putamen in the
   brain sections near the injection point in the Group B mice (Figures
   [121]6C,D) compared with the Group A mice (Figures [122]6A,B). However,
   there were also large regions of light staining and obvious
   infiltration of inflammatory cells in the Group C mice (Figures
   [123]6E,F). HE staining and light microscopy also revealed massive
   necrosis in the brain tissues of the Group C mice, and the brain cells
   showed various types of degeneration, including cytolysis, nuclear
   fragmentation, and indistinct boundaries between the cells. Notably,
   exfoliated vascular endothelial cells were also observed (Figures
   [124]6E,F). The degree of brain tissue necrosis was significantly
   higher in the Group C mice than in the Group B mice, and the normal
   structure of the brain tissue was lost or slightly disordered (Figures
   [125]6E,F).

Figure 6.

   [126]Figure 6
   [127]Open in a new tab

   Histopathological examination of mouse brain tissues 6 h after
   injection. Hematoxylin-and-eosin staining of the caudate putamen in
   brain tissues from blank control mice (A,B), control mice injected
   non-specific peptide (C,D), and mice injected with the mimic ZCP
   peptide (E,F). Bars: 100 μm.

   To identify the apoptotic cells after treatment, the presence of
   cleaved caspase 3 in the tissues was detected with immunohistochemical
   techniques. In addition to the massive necrosis induced by the mimic
   peptide in the Group C mice, the number of apoptotic cells was
   increased, as revealed by immunostaining with an antibody directed
   against cleaved caspase 3 (Figures [128]7E,F). However, rare apoptotic
   cells were observed in the untreated mice (Group A; Figures [129]7A,B)
   and the non-specific-peptide-treated mice (Group B; Figures [130]7C,D).

Figure 7.

   [131]Figure 7
   [132]Open in a new tab

   Immunohistochemical analysis of apoptotic cells in tissues after
   injection. Immunostaining for cleaved caspase 3 in brain tissues from
   blank control mice (A,B), control mice injected with non-specific
   peptide (C,D), and mice injected with synthetic ZCP mimic peptide
   (E,F). Apoptosis increased dramatically in the
   synthetic-peptide-treated mouse brains. Arrow indicates cells positive
   for cleaved caspase 3. Bars: 100 μm.

Discussion

   ZIKV infection is an emerging threat around the world and appears to be
   associated with brain diseases such as microcephaly in infants (Calvet
   et al., [133]2016; Cauchemez et al., [134]2016; Mlakar et al.,
   [135]2016; Rasmussen et al., [136]2016; Gabriel et al., [137]2017) and
   with Guillain–Barré syndrome (Brasil et al., [138]2016; Cao-Lormeau et
   al., [139]2016). A major concern associated with ZIKV infection is the
   apparent increased incidence of microcephaly in neonates born to
   ZIKV-infected mothers in Brazil (Calvet et al., [140]2016; Rubin et
   al., [141]2016). No specific antiviral medications or treatments are
   currently approved for ZVD because there has been little research into
   the molecular mechanisms underlying the potential association between
   ZIKV infection and brain diseases (Adibi et al., [142]2016; Bayer et
   al., [143]2016; Gabriel et al., [144]2016; Lazear et al., [145]2016;
   Nowakowski et al., [146]2016; Tang et al., [147]2016). Here, we created
   a possible genetic regulatory network for the proteins involved in the
   ZIKV–human interaction and microcephaly-associated proteins (Figures
   [148]S1–[149]S3 and Tables [150]S1, [151]S2). Systemic virology was
   used to generate this landscape map, which assumes that proteins within
   common signaling pathways are associated with the same upstream
   transcription factors (Figures [152]1A,B and Figure [153]S4).

   We found that the cell death signaling pathway is associated with both
   ZIKV infection and microcephaly and that P53 is the hub of the
   transcriptional regulatory network connecting ZIKV infection and
   microcephaly (Figure [154]2 and Figure [155]S5). Therefore, we inferred
   that ZIKV-related proteins might overlap the microcephaly-associated
   proteins at the P53 signaling pathway, where they probably interact
   with the MDM2 protein, inducing viral-infection-induced cell death,
   thereby leading to a disorder characterized by abnormal brain
   development (Figure [156]3A). In accordance with the findings of
   earlier studies (Yang et al., [157]2008; Bhuvanakantham et al.,
   [158]2010; Sips et al., [159]2012), the results of our molecular
   docking analysis suggest that the C-terminus of ZCP interacts with MDM2
   via the hydrophobic interactions that contribute to the ZCP–MDM2
   interactions (Figures [160]3B–E and Figure [161]S6). The similarity
   between the ZCP–MDM2 interaction and the P53–MDM2 interaction suggests
   that ZCP blocks the formation of the MDM2–P53 complex, causing P53
   release (Figure [162]4). When the MDM2-mediated degradation of P53 is
   prevented during brain development, the P53-mediated cell death pathway
   is activated, triggering the apoptosis of neural cells. To investigate
   whether ZCP is involved in cell death in the brain, we co-incubated a
   synthetic ZCP peptide with U87 cells (Figure [163]5) and injected them
   directly into the brains of Balb/C mice (Figures [164]6, [165]7 and
   Figure [166]S8). The results of both in vitro and in vivo experiments
   demonstrated that the synthetic ZCP mimic induced the death of U87
   cells and cells in the brains of mice. This was evident as changes in
   the morphological features of the cells and the presence of fluorescent
   staining characteristic of apoptosis, and the brain injury in the mice
   was also detected histopathologically. Understanding the molecular
   response to ZIKV infection will facilitate the discovery of clinically
   useful drugs for the treatment of ZVD and will accelerate the
   development of vaccines for the prevention of ZIKV infection
   (Kostyuchenko et al., [167]2016; Sirohi et al., [168]2016; Song et al.,
   [169]2016).

Materials and methods

Constructing a genetic regulatory network

   The integrated regulatory network was constructed based on a systematic
   integration of various high-throughput datasets. The transcriptional
   factors associated with the interaction genes were selected from the
   Transcriptional Regulatory Element Database (TRED). The integrated
   target genes–transcriptional factors regulatory network was constructed
   by using Cytoscape software v3.2.1 (cytoscape.org; Shannon et al.,
   [170]2003), which is an open-source software for visualizing complex
   networks and integrating these networks with any type of attribute
   data.

Systems analysis of gene ontology and biological pathways

   To map each interaction protein into a known phenotype, we used the
   databases Online Mendelian Inheritance in Man (OMIM) and Genome-wide
   association studies (GWAS), which are comprehensive, authoritative
   compendiums of human genes and genetic phenotypes with their functional
   annotations and disease classifications. To assign enriched gene
   ontology (GO) terms for host factors, DAVID Bioinformatics Resources
   v6.7 ([171]https://david.ncifcrf.gov/home.jsp) was used to query the
   list of identified host factors that were common to two or more
   studies. Molecular functions, biological processes, and cellular
   components that were identified with a confidence level of at least 95%
   were included in the analysis. For each topological module, a
   pathway-enrichment analysis of the biological pathways in which the
   module-related genes are believed to be involved was performed using
   DAVID online analytical tools. The pathway databases used in this study
   were KEGG ([172]www.genome.jp/kegg), REACTOME ([173]www.reactome.org),
   PANTHER ([174]www.pantherdb.org), and BIOCARTA ([175]www.biocarta.com)
   (Kanehisa et al., [176]2016; Mi et al., [177]2016).

Molecular structure preparation and molecular docking

   We used the ZIKV Capsid structure described in a previous publication
   (Ekins et al., [178]2016), which indicated that the modeled structure
   was reliable. We first performed a molecular dynamics (MD) simulation
   of the reported Capsid protein structures to be involved in the system
   composed of water and ions to obtain a stable three-dimensional
   structure of the ZIKV Capsid protein (see the detailed information in
   Data Sheet [179]1 as the Supplementary Materials). The system was
   equilibrated with an 80 ns MD simulation and the result of RMSD
   (Root-mean-square deviation) showed that the structure became
   convergent after 43 ns with a fluctuation range of 0.5–2.5 Å, probably
   during the 80 ns MD simulation (Figure [180]S6). Next, we employed the
   stable structure of ZCP as the receptor to dock with the MDM2 protein.
   All molecular structure visualizations were performed with PyMOL
   (Version 1.7.2, [181]http://pymol.org). In this study, AutoDock
   software version 4.2 ([182]http://autodock.scripps.edu) (Morris et al.,
   [183]2009) was employed to perform molecular docking as previously
   described for providing predictions of bound conformations. The ADT
   1.5.6 (Auto Dock Tools) software was employed to prepare the docking
   procedures. The key residues for ZIKV Capsid structure were within the
   C-terminal of the Capsid. This region contains amino acid residues
   65–97, and the number of runs was set to 100 in the molecular docking
   analysis. The MD simulation results were used to initiate the binding
   free energies calculations.

Cell culture

   U87 cells were grown at 37°C in a 5% CO[2] atmosphere in Dulbecco's
   modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum
   (Hyclone, USA). The medium was replaced every other day. Two synthetic
   peptide of the c-terminal sequence of ZIKV Capsid protein (Mimic
   peptide, residues 74–97, KKEAMEIIKKFKKDLAAMLRIINARRRRRRRR; Non-specific
   peptide, residues 30–53, LKRLPAGLLLGHGPIRMVLAILAFRRRRRRRR; Bankpeptide
   Co., Ltd., Hefei, China) was introduced to the U87 cells at a final
   concentration of 5 or 10 μM (ATCC, USA). The different experimental
   groups were cultured in six-well plates, with three replicates per
   group and each well contained 5 × 10^5 cells. Cell morphological
   changes were observed by microscopy. Each experiment was repeated three
   times using cells from three different cell preparations.

Cell viability

   The Cell viability of U87 cells was determined using CCK-8 assay
   (Dojindo). U87 cells at the density of 1 × 10^4 cells per well/100 μL
   were seeded in 96-well plate and cultured for 24 h to allow adherence
   and growth. After this time, cells were incubated with synthetic
   peptide using 0, 5, 10 μM doses for 4 h. After the treatment, cells
   were exposed to CCK-8 according to the manufacturer's instructions. The
   absorbance per well was measured at 540 nm using the POLARstar Omega
   Microplate Reader (BMG Labtech). Relative cell survival was expressed
   relative to the non-treated controls.

Tunel assay

   In Situ Cell Death Detection Kit, Fluorescein was obtained from
   Sigma-Aldrich. Staining was performed following the manufacturer's
   instruction. Cultured U87 cells were treated with mimic and
   non-specific peptide separately at the doses as labeled in figures.
   After 4 h peptide treatment, cells were fixed by 4% PFA
   Paraformaldehyde, PFA in PBS for 1 h at the room temperature. Cells
   were permeabilized using 0.1% Triton X-100 solution for 2 min on ice
   followed by twice rinsing by PBS. After the twice wash, cells were
   incubated with TUNEL reaction mixture for 1 h at 37°C in a humidified
   atmosphere in the dark. Cell nucleus was counterstained by DAPI. After
   PBS wash, the signal in cell nucleus was analyzed under fluorescent
   microscopy.

Apoptosis array and immunoblotting

   The effect of synthetic peptide to U87 cells was determined using the
   Human Apoptosis Array Kit from R&D Systems according to the
   manufacturer's instructions. Briefly, 2 × 10^6 U87 cells per well/2 ML
   were seeded in 6-well plate 24 h before treatment. Cell lysate was
   harvested followed by treating with the synthetic peptide at the
   concentration of 0 and 10 μM for 2 h. Cell lysates were mixed with a
   cocktail of biotinylated detection antibodies. Incubated with the array
   membrane by the sample/antibody mixture. After a wash to remove unbound
   material, streptavidin-HRP and chemiluminescent detection reagents were
   added. Immunoblotting was performed according with standard protocols.
   The cells were lysed with 1% SDS lysis buffer, and the protein
   concentration was determined using a BCA Assay Kit (Pierce). Protein
   samples were resolved on SDS-PAGE and then were transferred to PVDF
   membranes. The membranes were blocked with 5% BSA and then were
   incubated with primary and secondary antibodies, and then washed.
   Protein expression was determined by using ECL Detection Reagent
   (Pierce). Antibodies against cleaved caspase-3, cleaved caspase-9 come
   from Cell Signaling Technology. Anti-α-tubulin was obtained from Sigma.
   Anti p53 was obtained from Santa Cruze.

Elisa

   MDM2 concentration was determined using human total MDM2/HDM2 DuoSet IC
   ELISA kit (R&D Systems) according to manufacturer's instructions. 2 ×
   10^6 U87 cells per well/2 ML were seeded in 6-well plate. After the 24
   h culture, cells were treated with the synthetic peptide at the
   concentration of 0, 2.5, 5, 10, 20, 40, 80 μM doses for 2 h. Cell
   lysates were harvested and incubated to the prepared 96-well assay
   plate. Detection antibody and diluted Streptaviding-HRP was added
   subsequently. The colorimetric substrate was added and developed. The
   absorbance value per well was determined at 540 nm using the POLARstar
   Omega Microplate Reader (BMG Labtech). A standard curve was generated
   from plotting the absorbance for each concentration of standard sample
   vs. the corresponding concentration. Sample otal protein concentration
   was determined by BCA assay (Pierce).

Animals

   Female BALB/C mice weighing 20–22 g were maintained for more than 1
   week before commencing the experiment. The animals were housed in the
   animal facilities at Beijing Institute of Pharmacology and Toxicology.
   Animals received care according to the Division of Laboratory Animal
   Medicine guidelines, which were approved by the Association for
   Assessment and Accreditation of Laboratory Animal Care. This study was
   approved by the Committee on Animal Ethics in the Care and Use of
   Laboratory Animals of the Beijing Institute of Transfusion Medicine.
   All procedures were performed under general (Ethel) anesthesia using
   sterile surgical techniques.

Injection studies and histopathologic analysis

   Female BALB/C mice were randomly divided into three experimental groups
   with 10 mice in each group. Group A mice were used as a blank control;
   group B mice were injected with 5 μl of physiological saline containing
   0.25 mg of non-specific peptide per mouse; group C mice were injected
   with 5 μl of physiologic saline containing 0.25 mg of the synthetic
   mimic peptide per mouse. After injection, all mice were monitored and
   housed in the animal facilities. Brain specimens were obtained at the
   specified time points and processed for histological examination.
   Paraffin-embedded sections (4 mm) were stained with HE.
   Paraffin-embedded sections (4 mm) were deparaffinized through graded
   ethanol solutions. After an antigen retrieval procedure of 30 min using
   target retrieval solution (DAKO), the sections were stained with the
   antibody of cleaved caspase-3 (Cell Signaling Technology) using the
   avidin-biotin complex system (Vector Laboratory). DAB was used as the
   substrate. Cell nuclei were counterstained with haematoxylin.

Author contributions

   YuT, XG, SfL, SxL, YJ., TH, BL, XA, and ZM characterized the materials
   and designed the experiments, under the supervision of DF and YiT, and
   PZ implemented the experiments under the supervision of DF and YuT
   wrote the manuscript with further contributions from and DF and YiT
   analyzed the data. All authors reviewed the manuscript.

Conflict of interest statement

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgments