Edited by: Juliano Bordignon, Fundação Oswaldo Cruz (Fiocruz), Brazil
Reviewed by: Florencia Meyer, Mississippi State University, United States; Yhojan Rodríguez, Universidad del Rosario, Colombia
This article was submitted to Virology, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The first human Zika virus (ZIKV) outbreak was reported in Micronesia in 2007, followed by one in Brazil in 2015. Recent studies have reported cases in Europe, Oceania and Latin America. In 2016, ZIKV transmission was also reported in the US and the World Health Organization declared it a Public Health Emergency of International Concern. Because various neurological conditions are associated with ZIKV, such as microcephaly, Guillain-Barré syndrome, and other disorders of both the central and peripheral nervous systems, including encephalopathy, (meningo)encephalitis and myelitis, and because of the lack of reliable patient diagnosis, numerous ongoing studies seek to understand molecular mechanisms underlying ZIKV pathogenesis. Astrocytes are one of the most abundant cells in the CNS. They control axonal guidance, synaptic signaling, neurotransmitter trafficking and maintenance of neurons, and are targeted by ZIKV. In this study, we used a newly developed multiplexed aptamer-based technique (SOMAScan) to examine > 1300 human astrocyte cell proteins. We identified almost 300 astrocyte proteins significantly dysregulated by ZIKV infection that span diverse functions and signaling pathways, including protein translation, synaptic control, cell migration and differentiation.
More than 3 billion people are at risk for infection by arthropod-borne viruses (arboviruses). Zika virus (ZIKV) is a newly re-emerging mosquito-borne virus that is known to cause rare but serious birth defects (
The serious brain malformations in newborn babies infected with ZIKV
Since viruses are obligate intracellular parasites, they depend upon host cells and processes for replication. Numerous studies have demonstrated profound virus-induced alterations in the transcriptomic and proteomic profiles of virus-infected cells and organisms (for examples:
Human U-251 glioblastoma astrocytoma [U-251 MG (formerly known as U-373 MG; European Collection of Authenticated Cell Cultures – ECACC 09063001)] were cultured in Dulbecco’s modified Eagle’s/Nutrient Mixture F-12 (DMEM/F-12) medium. Monkey Vero cells (ATCC® # CCL-81TM) were cultured in Dulbecco’s modified Eagle’s medium (DMEM). Both media were supplemented with 10% Fetal Bovine Serum (FBS), non-essential amino acids, 2 mM
The Asian strain of ZIKV was a gift from Dr. David Safronetz, Chief of Special Pathogens, the National Microbiology Laboratory, Public Health Agency of Canada. Virus was propagated in Vero cells in DMEM supplemented with 2.5% FBS, non-essential amino acids, 2 mM
Vero cells were grown to 60–70% confluency in fully supplemented DMEM media and infected with ZIKV at MOI ∼ 0.001 PFU/cell. Media were replaced after virus adsorption with DMEM supplemented as above but containing 2.5% FBS. Supernatants were collected at different time-points to harvest virus, centrifuged at 600 ×
Vero cells were grown to 60–70% confluency in 6-well plates. Serial 10-fold dilutions of viral stocks were made in gel saline [137 mM NaCl, 0.2 mM CaCl2, 0.8 mM MgCl2, 19 mM HBO3, 0.1 mM Na2B4O7, 0.3% (wt/vol) gelatin], and adsorbed in duplicate on the Vero cell monolayers. After viral attachment (2 h in a 37°C CO2 incubator with periodic rocking), 2.5 ml per well of a 50:50 (vol/vol) mixture of [1.2% Type I Agarose (Difco Laboratories, Detroit, Mich.)] and [2 × completed Medium 199 (Medium 199 with 6% FBS, 4 mM glutamine, 10 μg of gentamicin sulfate per ml, and 3 μg of amphotericin B per ml)] was added to each well to overlay cells. Cells were incubated at 37°C for 5 days and then overlaid with 1% Bacto Agar in 1× PBS that contained 0.04% neutral red. Plaques were counted 18–21 h after neutral red staining.
U-251 cells were grown to 60-70% confluency in fully supplemented DMEM/F-12 media and infected at MOI = 3, which, by Poisson calculation, suggests > 95% of cells are infected. Virus was adsorbed in gel saline as described above for 2 h in a 37°C CO2 incubator with periodic rocking. Mock-infected cells were treated similarly in gel saline that contained no virus. After adsorption, cells were overlaid with DMEM/F12 supplemented as described above but containing 2.5% FBS and incubated at 37°C for various periods of time as indicated below.
Infected- and mock-treated U-251 cells were microscopically observed for cytopathic effect (CPE) at 6, 12, 24, 48, and 72 h post-infection (hpi) using a Nikon TE-2000 inverted microscope. All images were taken with a Canon A-700 digital camera and exported into PowerPoint for minimal adjustment of brightness and contrast.
U-251 cells were seeded on spotted slides at 20,000 cells/spot, infected at an MOI = 3 as described above and processed at 12, 24, and 48 hpi. Cells were fixed using 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 in PBS for 5 min. Cells were then blocked in 3% BSA blocking solution for 90 min, followed by overnight incubation at 4°C with mouse anti-ZIKV NS1 primary antibody (BioFront Technologies; cat # BF-1225-06) in 1% BSA and PBS. Cells were washed with PBT buffer (PBS with 0.2% Tween-20) and secondary anti-mouse antibody tagged with Alexa FluorTM 488 (Invitrogen #A-11001) was added for 1 h. Slides were washed 3× with PBT buffer followed by mounting using DAPI-Prolong® Gold AntiFade to stain nuclei. Slide edges were sealed using nail polish and the slides kept at 4°C. until visualized. Images were taken with a Zeiss Axio Observer ZI inverted microscope using 20× objective. AxioVision 4.8.2 software was used to capture and edit the images before exporting them.
Cell viability was measured using the WST-1 assay according to manufacturer’s instructions (Roche), but adding 8 μL of WST-1 reagent instead of 10 μL into each well of a 96-well plate at desired times. The cells were incubated for 2 h at 37°C and then absorbance was measured at 440 and 610 nm. Absorbance at 610 nm was subtracted from that at 440 nm and the values of the ZIKV-infected samples were normalized with their respective mock samples at each time point. A minimum of triplicate experiments were analyzed.
Mock- and ZIKV-infected U-251 samples were harvested at each of various time points (12, 24, and 48 hpi) and washed 3× with >50-volumes of PBS to remove media and culture FBS. Washed cells were lysed with MPER® (Pierce; Rockford, IL, United States) supplemented with 1× HALT® Protease inhibitor (Pierce; Rockford, IL, United States). After cell lysis, lysates were centrifuged at 14,000 ×
Protein concentrations of BCA-determined cell lysates were adjusted to 200 ng/μL, and 70 μL of each sample submitted for SOMAScan® analysis in-house on a SomaLogics®-licensed platform in the Manitoba Centre for Proteomics and Systems Biology as described (
BCA-quantified protein samples were adjusted to load 10 μg of protein per gel lane. Samples were heated to 95°C for 5 min and resolved by mini-10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) until the loading dye had just run off the bottom edge of the gel. The proteins were transferred to PVDF [Immobilon-P polyvinylidene difluoride membrane (Millipore)] for 2 h in ice-cold buffer, followed by overnight blocking of the membrane in 5% skim milk in 1× TBST. Primary antibodies were added to each blot at 1:1000 dilution in 1% milk/TBST overnight. Primary antibodies used were: mouse monoclonal anti-ZIKV NS1 (BioFront Technologies # BF-1225-06); anti-ZIKV NS3 (GeneTex Inc. # GTX133309), anti-ZIKV envelope protein (GeneTex Inc. # GTX133314), anti-STAT3 (Cell Signaling # 124H6), anti-β-actin (Cell Signaling # 8H10D10); rabbit polyclonal anti-fibronectin (Abcam # ab2413), anti-SPARC (Cell Signaling # 5420S), and anti-cystatin C (Abcam # ab109508). After overnight binding, membranes were washed 3× with TBST and appropriate goat HRP-conjugated anti-rabbit (Cell Signaling # 7074) or anti-mouse (Cell Signaling # 7076) secondary antibodies were added for 1 h. The blots were washed 3 additional times with 1× TBST, developed with ECL western blotting peroxidase substrate for chemiluminescence and imaged with an enhanced chemiluminescence (ECL) detection machine (Amersham-Pharmacia Biotech); ImageJ was used to analyze each blot and each band in each blot was normalized to its respective actin control and to its time-matched mock-infected band intensity.
Relative fluorescent units values for each of the 1322 analytes (1307 unique proteins + 15 internal SOMA control analytes) in each of 3 biologic replicates, each consisting of a ZIKV-infected sample and a time-matched non-infected mock sample at 12, 24, and 48 hpi were imported into Excel and converted to Log2 values. Fold-changes were determined for each of the nine infected samples compared to their time-matched mock samples. The fold-changes were analyzed for significance by both Students
Unlike the mock-treated astrocytic U-251 cells, ZIKV-infected astrocytes show a visible cytopathic effect (CPE) over time as they start to circularize and lift from the surface (
Zika virus (ZIKV) productively infects U-251 astrocyte cells.
We analyzed the cellular proteome of ZIKV-infected U-251 astrocytes at 12, 24, and 48 hpi and compared them to time-matched mock-treated cells using the novel SOMAScan platform. A total of 296 cellular proteins were significantly over- or under-expressed compared to mock, non-infected, with 17 over-expressed and 17 under-expressed at 12 hpi, 23 over- and 40 under-expressed at 24 hpi, and 45 over- and 209 under-expressed at 48 hpi (
Numbers of significantly dysregulated ZIKV-infected U-251 proteins.
Number that are significant | Total Unique | 12 hpi | 24 hpi | 48 hpi |
---|---|---|---|---|
and fold-change > 1.000 | 296 | 17 | 23 | 45 |
and fold-change < 0.9999 | 17 | 40 | 209 | |
and fold-change > 1.100 | 272 | 2 | 21 | 43 |
and fold-change < 0.9091 | 8 | 40 | 209 | |
and fold-change > 1.250 | 244 | 0 | 10 | 30 |
and fold-change < 0.8000 | 3 | 37 | 204 | |
and fold-change > 1.500 | |
|
|
|
and fold-change < 0.6667 | |
|||
and fold-change > 1.750 | 113 | 0 | 1 | 9 |
and fold-change < 0.5714 | 0 | 4 | 102 | |
and fold-change > 2.000 | 80 | 0 | 1 | 7 |
and fold-change < 0.5000 | 0 | 2 | 73 | |
Global dysregulation of U-251 proteins and cellular processes.
U-251 proteins significantly dysregulated by ZIKV infection.
12 hpi |
24 hpi |
48 hpi | |||||
---|---|---|---|---|---|---|---|
Entrez Gene Symbol | Protein | Fold Change | Fold Change | Fold Change | |||
CXCL11 | C-X-C motif chemokine 11 | 0.865 | |||||
NACA | Nascent polypeptide-associated complex subunit alpha | 1.040 | 0.281 | 0.974 | 0.761 | ||
CCL5 | C-C motif chemokine 5 | 1.041 | 0.178 | 1.316 | |||
CKB | Creatine kinase B-type | 0.989 | 0.860 | 0.988 | 0.931 | ||
SSRP1 | FACT complex subunit SSRP1 | 0.995 | 0.968 | 1.422 | |||
ISG15 | Ubiquitin-like protein ISG15 | 0.998 | 0.982 | 1.261 | |||
HMGN1 | Non-histone chromosomal protein HMG-14 | 1.016 | 0.815 | 1.169 | 0.143 | ||
NUDCD3 | NudC domain-containing protein 3 | 0.986 | 0.738 | 1.135 | 0.125 | ||
PLCG1 | 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 | 1.015 | 0.775 | 1.183 | |||
METAP2 | Methionine aminopeptidase 2 | 0.992 | 0.947 | 1.118 | 0.076 | ||
ADSL | Adenylosuccinate lyase | 1.094 | 0.101 | 1.192 | 0.143 | ||
PARK7 | Protein deglycase DJ-1 | 1.028 | 0.326 | 1.225 | 0.057 | ||
HSPA1A | Heat shock 70 kDa protein 1A | 1.160 | 1.224 | ||||
GDI2 | Rab GDP dissociation inhibitor beta | 1.065 | 0.512 | 1.114 | 0.111 | ||
PEBP1 | Phosphatidylethanolamine-binding protein 1 | 0.993 | 0.942 | 1.169 | 0.189 | ||
UBE2N | Ubiquitin-conjugating enzyme E2 N | 1.047 | 0.585 | 1.101 | 0.197 | ||
PPA1 | Inorganic pyrophosphatase | 1.035 | 0.684 | 1.139 | 0.075 | ||
IFNL1 | Interferon lambda-1 | 0.908 | 0.060 | 1.239 | |||
EIF5A | Eukaryotic translation initiation factor 5A-1 | 1.040 | 0.653 | 1.143 | |||
STC1 | Stanniocalcin-1 | 0.902 | 0.437 | ||||
CST3 | Cystatin-C | 0.913 | 0.445 | ||||
IGFBP2 | Insulin-like growth factor-binding protein 2 | 0.976 | 0.689 | ||||
SFRP1 | Secreted frizzled-related protein 1 | 0.904 | 0.211 | ||||
APP | Amyloid beta A4 protein | 0.976 | 0.786 | ||||
MFGE8 | Lactadherin | 0.946 | 0.410 | ||||
MDK | Midkine | 1.019 | 0.629 | ||||
IGFBP7 | Insulin-like growth factor-binding protein 7 | 0.919 | 0.256 | ||||
IGFBP5 | Insulin-like growth factor-binding protein 5 | 0.982 | 0.767 | ||||
CTSA | Lysosomal protective protein | 0.946 | 0.353 | ||||
LRIG3 | Leucine-rich repeats and immunoglobulin-like domains protein 3 | 0.978 | 0.688 | ||||
FGFR1 | Fibroblast growth factor receptor 1 | 0.937 | 0.291 | ||||
PCSK9 | Proprotein convertase subtilisin/kexin type 9 | 1.012 | 0.840 | ||||
CFI | Complement factor I | 0.911 | 0.342 | ||||
TIMP2 | Metalloproteinase inhibitor 2 | 0.999 | 0.986 | ||||
FSTL1 | Follistatin-related protein 1 | 1.012 | 0.769 | ||||
GRN | Granulins | 0.961 | 0.578 | ||||
HS6ST1 | Heparan-sulfate 6- |
0.964 | 0.241 | 0.710 | |||
C4A C4B | Complement C4 | 0.916 | 0.265 | 1.044 | 0.711 | ||
PLAU | Urokinase-type plasminogen activator | 0.972 | 0.530 | 0.779 | |||
TNC | Tenascin | 0.973 | 0.480 | 0.732 | |||
GPNMB | Transmembrane glycoprotein NMB | 1.014 | 0.797 | 0.914 | 0.419 | ||
GFRA1 | GDNF family receptor alpha-1 | 0.971 | 0.638 | 0.744 | |||
LAMA1 LAMB1 LAMC1 | Laminin | 0.949 | 0.319 | 0.762 | |||
THBS2 | Thrombospondin-2 | 0.930 | 0.133 | 0.710 | |||
ACVR1B | Activin receptor type-1B | 1.001 | 0.990 | 1.142 | 0.194 | ||
XRCC6 | X-ray repair cross-complementing protein 6 | 0.936 | 0.222 | 0.906 | 0.357 | ||
GNS | 1.004 | 0.948 | 0.878 | 0.179 | |||
CCL13 | C-C motif chemokine 13 | 1.003 | 0.958 | 1.084 | 0.331 | ||
CCL20 | C-C motif chemokine 20 | 0.969 | 0.686 | 1.100 | 0.421 | ||
L1CAM | Neural cell adhesion molecule L1 | 0.900 | 0.187 | 0.880 | 0.070 | ||
IGF1R | Insulin-like growth factor 1 receptor | 1.018 | 0.624 | 0.972 | 0.669 | ||
ICAM5 | Intercellular adhesion molecule 5 | 0.971 | 0.494 | 0.939 | 0.428 | ||
PLXNB2 | Plexin-B2 | 0.974 | 0.604 | 0.826 | |||
GSN | Gelsolin | 0.913 | 0.312 | 0.899 | 0.294 | ||
C4A C4B | Complement C4b | 0.958 | 0.570 | 1.115 | 0.336 | ||
IL17RA | Interleukin-17 receptor A | 0.951 | 0.541 | 0.930 | 0.445 | ||
LYN | Tyrosine-protein kinase Lyn, isoform B | 0.956 | 0.635 | 0.904 | 0.450 | ||
PF4 | Platelet factor 4 | 0.981 | 0.702 | 1.118 | 0.192 | ||
PROK1 | Prokineticin-1 | 0.986 | 0.806 | 1.139 | 0.136 | ||
HIST1H1C | Histone H1.2 | 0.923 | 0.289 | 0.821 | 0.179 | ||
RTN4R | Reticulon-4 receptor | 0.993 | 0.772 | 0.906 | 0.319 | ||
UNC5C | Netrin receptor UNC5C | 1.004 | 0.875 | 0.837 | 0.086 | ||
LIFR | Leukemia inhibitory factor receptor | 0.955 | 0.593 | 0.686 | |||
LYN | Tyrosine-protein kinase Lyn | 0.925 | 0.387 | 0.897 | 0.429 | ||
FGF12 | Fibroblast growth factor 12 | 0.981 | 0.709 | 1.061 | 0.512 | ||
HSD17B10 | 3-hydroxyacyl-CoA dehydrogenase type-2 | 0.984 | 0.688 | 0.991 | 0.917 | ||
CMA1 | Chymase | 0.989 | 0.804 | 1.126 | 0.141 | ||
CRISP3 | Cysteine-rich secretory protein 3 | 0.994 | 0.934 | 1.122 | 0.208 | ||
ALB | Serum albumin | 0.998 | 0.965 | 1.104 | 0.295 | ||
TNFRSF1A | Tumor necrosis factor receptor superfamily member 1A | 1.005 | 0.904 | 0.783 | |||
SRC | Proto-oncogene tyrosine-protein kinase Src | 0.977 | 0.706 | 0.907 | 0.353 | ||
FER | Tyrosine-protein kinase Fer | 0.982 | 0.790 | 1.226 | 0.527 | ||
TFPI | Tissue factor pathway inhibitor | 0.948 | 0.216 | 0.715 | |||
DYNLL1 | Dynein light chain 1, cytoplasmic | 0.990 | 0.823 | 1.095 | 0.308 | ||
MMP2 | 72 kDa type IV collagenase | 0.983 | 0.729 | 0.877 | 0.103 | ||
RNASEH1 | Ribonuclease H1 | 0.976 | 0.709 | 1.006 | 0.957 | ||
FN1 | Fibronectin | 1.013 | 0.750 | 0.851 | 0.075 | ||
SERPINA3 | Alpha-1-antichymotrypsin | 0.945 | 0.176 | 1.053 | 0.596 | ||
NRXN3 | Neurexin-3-beta | 0.983 | 0.479 | 0.918 | 0.281 | ||
PON1 | Serum paraoxonase/arylesterase 1 | 0.971 | 0.598 | 1.098 | 0.347 | ||
FSTL3 | Follistatin-related protein 3 | 0.910 | 0.370 | 0.727 | |||
NID1 | Nidogen-1 | 0.977 | 0.641 | 0.843 | 0.066 | ||
B2M | Beta-2-microglobulin | 0.969 | 0.580 | 0.721 | |||
UNC5D | Netrin receptor UNC5D | 0.935 | 0.362 | 0.869 | 0.239 | ||
AGER | Advanced glycosylation end product-specific receptor, soluble | 1.012 | 0.854 | 1.092 | 0.351 | ||
MICB | MHC class I polypeptide-related sequence B | 1.009 | 0.875 | 0.978 | 0.774 | ||
ECM1 | Extracellular matrix protein 1 | 0.981 | 0.655 | 0.763 | |||
LY9 | T-lymphocyte surface antigen Ly-9 | 1.009 | 0.925 | 1.139 | 0.080 | ||
ADAM12 | Disintegrin and metalloproteinase domain-containing protein 12 | 0.988 | 0.764 | 0.952 | 0.616 | ||
PTN | Pleiotrophin | 1.031 | 0.548 | 0.769 | |||
HIST1H3A | Histone H3.1 | 0.994 | 0.929 | 0.761 | |||
TF | Serotransferrin | 0.903 | 0.206 | 1.100 | 0.386 | ||
NOTCH1 | Neurogenic locus notch homolog protein 1 | 0.987 | 0.854 | 0.877 | 0.215 | ||
CDON | Cell adhesion molecule-related/down-regulated by oncogenes | 0.954 | 0.263 | 1.010 | 0.906 | ||
STAT3 | Signal transducer and activator of transcription 3 | 1.018 | 0.770 | 0.903 | 0.178 | ||
CTSV | Cathepsin L2 | 0.990 | 0.777 | 0.714 | |||
C5 | C5a anaphylatoxin | 1.008 | 0.903 | 1.122 | 0.172 | ||
KIF23 | Kinesin-like protein KIF23 | 0.930 | 0.182 | 0.884 | 0.066 | ||
BOC | Brother of CDO | 0.931 | 0.314 | 1.077 | 0.619 | ||
IL15RA | Interleukin-15 receptor subunit alpha | 0.992 | 0.862 | 1.137 | 0.169 | ||
GPC2 | Glypican-2 | 1.005 | 0.895 | 1.148 | 0.303 | ||
SERPINE2 | Glia-derived nexin | 0.998 | 0.967 | 1.058 | 0.500 | ||
ANGPT2 | Angiopoietin-2 | 0.971 | 0.501 | 1.137 | 0.136 | ||
F11 | Coagulation Factor XI | 1.006 | 0.899 | 1.110 | 0.235 | ||
CTSS | Cathepsin S | 1.007 | 0.894 | 1.093 | 0.259 | ||
COL18A1 | Endostatin | 0.992 | 0.759 | 0.781 | |||
PRKACA | cAMP-dependent protein kinase catalytic subunit alpha | 1.060 | 0.071 | 1.046 | 0.608 | ||
SPARC | SPARC | 1.092 | 0.116 | 0.867 | 0.105 | ||
PLAT | Tissue-type plasminogen activator | 0.992 | 0.780 | 0.885 | 0.145 | ||
PRKCA | Protein kinase C alpha type | 0.946 | 0.296 | 1.018 | 0.925 | ||
THBS1 | Thrombospondin-1 | 1.014 | 0.792 | 0.905 | 0.402 | ||
LGALS8 | Galectin-8 | 0.979 | 0.640 | 0.813 | |||
AKT1 AKT2 AKT3 | RAC-alpha/beta/gamma serine/threonine-protein kinase | 0.960 | 0.526 | 0.973 | 0.780 | ||
KLK13 | Kallikrein-13 | 1.011 | 0.833 | 1.111 | 0.200 | ||
POSTN | Periostin | 0.988 | 0.852 | 1.161 | 0.230 | ||
MRC2 | C-type mannose receptor 2 | 0.975 | 0.455 | 1.120 | 0.389 | ||
ICOS | Inducible T-cell costimulator | 1.010 | 0.849 | 1.099 | 0.305 | ||
PDK1 | [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 1, mitochondrial | 1.025 | 0.587 | 1.075 | 0.244 | ||
DKK1 | Dickkopf-related protein 1 | 0.916 | 0.748 | ||||
METAP1 | Methionine aminopeptidase 1 | 0.994 | 0.956 | 1.030 | 0.805 | ||
DDC | Aromatic- |
0.950 | 0.281 | 1.079 | 0.349 | ||
MAP2K3 | Dual specificity mitogen-activated protein kinase kinase 3 | 1.011 | 0.609 | 1.087 | 0.269 | ||
CAMK2D | Calcium/calmodulin-dependent protein kinase type II subunit delta | 0.997 | 0.963 | 1.086 | 0.577 | ||
MET | Hepatocyte growth factor receptor | 1.060 | 0.290 | 1.074 | 0.412 | ||
ALCAM | CD166 antigen | 1.030 | 0.540 | 0.973 | 0.574 | ||
EGFR | Epidermal growth factor receptor | 1.053 | 0.236 | 1.026 | 0.579 | ||
LGALS3BP | Galectin-3-binding protein | 0.983 | 0.816 | 0.826 | 0.120 | ||
TGFBI | Transforming growth factor-beta-induced protein ig-h3 | 1.016 | 0.720 | 0.836 | |||
PSMA2 | Proteasome subunit alpha type-2 | 0.988 | 0.782 | 1.035 | 0.732 | ||
CTSH | Cathepsin H | 1.000 | 0.992 | 1.071 | 0.425 | ||
EFNA2 | Ephrin-A2 | 1.036 | 0.195 | 0.984 | 0.841 | ||
CAMK2B | Calcium/calmodulin-dependent protein kinase type II subunit beta | 0.989 | 0.873 | 1.075 | 0.644 | ||
EPHA3 | Ephrin type-A receptor 3 | 1.005 | 0.914 | 1.065 | 0.356 | ||
FYN | Tyrosine-protein kinase Fyn | 0.970 | 0.492 | 1.030 | 0.643 | ||
TBP | TATA-box-binding protein | 0.986 | 0.819 | 0.977 | 0.797 | ||
EPHB2 | Ephrin type-B receptor 2 | 0.995 | 0.914 | 1.028 | 0.683 | ||
DKK4 | Dickkopf-related protein 4 | 0.897 | 0.053 | 0.779 | |||
CSK | Tyrosine-protein kinase CSK | 1.011 | 0.912 | 1.051 | 0.822 | ||
PLAUR | Urokinase plasminogen activator surface receptor | 0.988 | 0.735 | 0.925 | 0.277 | ||
ADRBK1 | beta-adrenergic receptor kinase 1 | 0.989 | 0.865 | 0.995 | 0.941 | ||
CTSB | Cathepsin B | 0.978 | 0.622 | 1.123 | 0.120 | ||
HSPB1 | Heat shock protein beta-1 | 1.010 | 0.819 | 1.007 | 0.951 | ||
CAMK2A | Calcium/calmodulin-dependent protein kinase type II subunit alpha | 0.972 | 0.547 | 1.120 | 0.528 | ||
MCL1 | Induced myeloid leukemia cell differentiation protein Mcl-1 | 0.984 | 0.679 | 0.941 | 0.401 | ||
ROBO2 | Roundabout homolog 2 | 1.044 | 0.164 | 0.973 | 0.659 | ||
MATN2 | Matrilin-2 | 0.992 | 0.825 | 1.037 | 0.649 | ||
CSRP3 | Cysteine and glycine-rich protein 3 | 0.990 | 0.888 | 0.902 | 0.271 | ||
PDE11A | Dual 3′,5′-cyclic-AMP and -GMP phosphodiesterase 11A | 1.022 | 0.716 | 0.997 | 0.920 | ||
TK1 | Thymidine kinase, cytosolic | 0.966 | 0.572 | 0.994 | 0.962 | ||
MAPK14 | Mitogen-activated protein kinase 14 | 0.985 | 0.799 | 1.016 | 0.874 | ||
NRP1 | Neuropilin-1 | 0.952 | 0.361 | 0.839 | 0.094 | ||
PIK3CA PIK3R1 | PIK3CA/PIK3R1 | 0.982 | 0.828 | 0.972 | 0.802 | ||
PDPK1 | 3-phosphoinositide-dependent protein kinase 1 | 0.967 | 0.638 | 1.031 | 0.807 | ||
APOA1 | Apolipoprotein A-I | 1.000 | 0.991 | 1.106 | 0.228 | ||
SNX4 | Sorting nexin-4 | 1.023 | 0.477 | 1.147 | 0.503 | ||
SLAMF7 | SLAM family member 7 | 1.030 | 0.498 | 1.081 | 0.264 | ||
PPP3CA PPP3R1 | Calcineurin | 0.982 | 0.760 | 1.005 | 0.951 | ||
PRTN3 | Myeloblastin | 0.988 | 0.785 | 1.093 | 0.296 | ||
CCL15 | C-C motif chemokine 15 | 1.000 | 0.996 | 1.121 | 0.205 | ||
GAS1 | Growth arrest-specific protein 1 | 1.012 | 0.675 | 1.007 | 0.917 | ||
IL6ST | Interleukin-6 receptor subunit beta | 1.021 | 0.468 | 0.907 | 0.241 | ||
LRPAP1 | alpha-2-macroglobulin receptor-associated protein | 1.047 | 0.084 | 0.991 | 0.794 | ||
LCN2 | Neutrophil gelatinase-associated lipocalin | 0.943 | 0.322 | 1.142 | 0.138 | ||
TNFRSF21 | Tumor necrosis factor receptor superfamily member 21 | 1.023 | 0.435 | 0.968 | 0.720 | ||
CXCL6 | C-X-C motif chemokine 6 | 1.029 | 0.448 | 0.975 | 0.788 | ||
MPL | Thrombopoietin Receptor | 0.970 | 0.481 | 1.111 | 0.276 | ||
COL23A1 | Collagen alpha-1(XXIII) chain | 0.971 | 0.563 | 1.150 | 0.216 | ||
NLGN4X | Neuroligin-4, X-linked | 0.978 | 0.587 | 1.022 | 0.822 | ||
SERPINE1 | Plasminogen activator inhibitor 1 | 1.028 | 0.781 | 1.141 | 0.341 | ||
CCDC80 | Coiled-coil domain-containing protein 80 | 1.063 | 0.145 | 0.969 | 0.689 | ||
To validate the SOMAScan results, we selected four cellular proteins predicted to have dysregulated expression by ZIKV infection and assessed their quantities at various times post infection. Fibronectin is involved in astrocytic cell growth, migration and actin polymerization, and STAT3 and SPARC are involved in astrocytic synaptic control. Commercial antibodies are available for each of these, and also for CST3 and were used to probe ZIKV-infected astrocytes (
A global overview of these proteins with altered levels of expression, using IPA, indicates that cell death functions are highly activated at 48 hpi, while cell survival, cell outgrowth, cell migration, cell spreading and cell-to-cell contact functions are inhibited at 48 hpi (
Lists of the ZIKV-induced differentially expressed U-251 proteins were uploaded to Ingenuity Pathway Analysis (IPA) for network analysis. Multiple networks that contain ≥ 12 focus molecules and with scores ≥ 20 were identified. The IPA-generated scores for each network are based on the fit between the network and that of the set of focus genes identified in that network; this score is a measure of likelihood of finding those identified genes in that particular network by random chance (
Significant differentially regulated U-251 protein networks. Proteins and their levels of regulation were imported into the Ingenuity Pathways Analysis (IPA®) tool and interacting pathways were constructed under default settings. Three of the top dysregulated U-251 cell networks that contain 12 or more “focus” molecules (molecules significantly up- or down-regulated) and that have network scores ≥ 20 at 48 hpi are identified. Red: significantly up-regulated proteins; pink: moderately up-regulated proteins; gray: proteins identified but not significantly regulated; light green: moderately down-regulated proteins; dark green: significantly down-regulated proteins; white: proteins known to be in network, but not covered within SOMAScan panel; dashed lines represent predicted or indirect interactions; solid lines represent direct known interactions. These networks, and top Vero cell differentially regulated networks, at 12, 24, and 48 hpi are shown in
We have previously reported SOMAScan® analyses of ZIKV-infected monkey Vero cells (
Six canonical pathways; Axonal guidance signaling, FGF signaling, Glioma Signaling, STAT3 signaling, ERK/MAPK and AMPK signaling, which are highly important in astrocytic functions, were predicted to be dysregulated by ZIKV infection by 48 hpi (
Dysregulated cellular molecules that potentially result in impairment of astrocytic functions crucial for astrocyte-mediated synaptic control, cell growth and migration. Differentially expressed proteins were imported into IPA and used to predict significant dysregulation of astrocyte/brain-specific functions.
U-251 axonal guidance proteins significantly dysregulated by ZIKV by 48 hpi.
Entrez Gene Symbol | Protein | Fold Change | Location | Type | |
---|---|---|---|---|---|
ADAM12 | ADAM metallopeptidase domain 12 | 0.49 | 0.004 | Plasma Membrane | Peptidase |
EFNA2 | Ephrin A2 | 0.60 | 0.010 | Plasma Membrane | Kinase |
EPHA3 | EPH receptor A3 | 0.60 | 0.016 | Plasma Membrane | Kinase |
EPHB2 | EPH receptor B2 | 0.60 | 0.016 | Plasma Membrane | Kinase |
FGFR1 | Fibroblast growth factor receptor 1 | 0.20 | 0.000 | Plasma Membrane | Kinase |
FYN | FYN proto-oncogene, Src family tyrosine kinase | 0.60 | 0.010 | Plasma Membrane | Kinase |
L1CAM | L1 cell adhesion molecule | 0.34 | 0.001 | Plasma Membrane | Other |
MET | MET proto-oncogene, receptor tyrosine kinase | 0.58 | 0.012 | Plasma Membrane | Kinase |
MMP2 | Matrix metallopeptidase 2 | 0.46 | 0.003 | Extracellular Space | Peptidase |
NRP1 | Neuropilin 1 | 0.64 | 0.018 | Plasma Membrane | Transmembrane receptor |
PLCG1 | Phospholipase C gamma 1 | 1.81 | 0.004 | Cytoplasm | Enzyme |
PLXNB2 | Plexin B2 | 0.34 | 0.001 | Plasma Membrane | Transmembrane receptor |
PRKACA | Protein kinase cAMP-activated catalytic subunit alpha | 0.54 | 0.002 | Cytoplasm | Kinase |
PRKCA | Protein kinase C alpha | 0.54 | 0.004 | Cytoplasm | Kinase |
PROK1 | Prokineticin 1 | 0.39 | 0.003 | Extracellular Space | Growth factor |
ROBO2 | Roundabout guidance receptor 2 | 0.63 | 0.011 | Plasma Membrane | Transmembrane receptor |
RTN4R | Reticulon 4 receptor | 0.39 | 0.002 | Plasma Membrane | Transmembrane receptor |
UNC5C | UNC-5 netrin receptor C | 0.39 | 0.002 | Plasma Membrane | Transmembrane receptor |
UNC5D | UNC-5 netrin receptor D | 0.49 | 0.002 | Plasma Membrane | Other |
Based on the over- and under-expression of a number of downstream molecules, 56 upstream molecules were identified and their activity was predicted to be significantly inhibited, and 46 upstream molecules were identified and predicted to be significantly activated by ZIKV by 48 hpi (
Dysregulation of upstream molecules that control downstream pathways, regulators, and gene expression.
We employed a novel proteomic approach to assess ZIKV-induced alterations in levels of U-251 astrocyte protein expression during a time course after infection. We showed that ZIKV can successfully infect and replicate in U-251 cells and that under our infection conditions (MOI = 3), >90% of the cells were infected by 48 hpi (
One of the dysregulated cellular systems was the Post-translational modification, protein degradation and synthesis network (
Another dysregulated cellular system was the Cellular assembly and organization, cellular function and maintenance and cellular development network. Molecules significantly affected in this network included kinases (PDPK1, EPHA3, EFNA2, PRKCA, and SRC), transmembrane receptors (GFRA1 and ROBO2) and other molecules (CSRP3, GDI2, GNS, GNS, ICAM5, and SSRP1) (
A third major dysregulated cellular system was the Cell development, cell growth, proliferation and cell cycle network. The molecules significantly affected in this network included kinases (CSK and EGFR), transmembrane receptors (ICOS, IL6ST, and MICB), translation regulator (EIF5A), cytokine (IFNL1) and other molecules (LY9, MATN2, SLAMF7, SNX4, and TF) (
In addition to astrocyte cell migration, reactivity and differentiation, IPA also predicted ZIKV impacting their interaction with neural cells (
IPA analyses also predicted activation/inhibition of various upstream regulators that could stall a variety of cellular canonical pathways (
The 26S proteasome complex was another upstream regulator predicted to be activated by ZIKV infection (
In conclusion, ZIKV successfully infects and replicates in astrocytic U-251 cells. A detailed proteomic analysis of the ZIKV-induced dysregulated cellular proteins revealed numerous dysregulated networks and cellular functions, especially those pertaining to astrocytic functions in the brain, and upstream regulator molecules that provide more explanation of how ZIKV can cause neurodegeneration and impairment of neural circuit and network development resulting in conditions like microcephaly. Extension of similar studies to other cell types infectable by ZIKV are warranted to develop a fuller comprehension of ZIKV-induced proteomic alterations that could be targeted for therapeutic intervention.
All datasets generated for this study are included in the manuscript and/or the
AS performed most of the experiments, interpreted the results, and wrote the manuscript first draft. KG assisted in experimental set-up, in results interpretation, and edited the manuscript. KC provided overall guidance, interpreted the results, and edited the manuscript.
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.
The Supplementary Material for this article can be found online at: