Cell lines
Human trophoblasts cells Bewo (ATCC, Cat. CCL-98), JEG-3 (ATCC, Cat. HTB-36), HTR-8 (ATCC, Cat. CRL-3271), and PHTs were cultured with Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, (DMEM/F-12 GIBCO, Cat. 11330032) supplemented with 10% fetal bovine serum (FBS) (Gibco, 16140071) and maintained at 37 °C with 5% CO2. THP-1 (ATCC, Cat. TIB-202) was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco, A1049101) supplemented with 10% FBS and 0.05 mM 2-Mercaptoethanol (Gibco, 21985023). THP-1 differentiation was performed as previously described99. PHTs were isolated from term placentas from deidentified uncomplicated pregnancies at Barnes-Jewish Hospital Labor and Delivery Service, St. Louis, MO. PHTs were thawed and cultured in a 6-well plate (Corning, 353046) at 2 x 106 cells/well using cell culture media described previously100. Following cell attachment, PHTs were washed with Iscove's Modified Dulbecco's Medium (IMDM; Gibco, 12440053) with 10% FBS, 100 U/mL Penicillin-Streptomycin (Gibco, 15070063) 10 μM Y-27632 (Selleckchem, S1049), 0.05 mM 2-Mercaptoethanol (Gibco, 21985023), and 30 ng/mL mouse EGF Recombinant Protein (Gibco, PMG8041). Cells were maintained in IMDM media for three days, subcultured at a 1:2 split ratio, and maintained for 5 days until virus infection in IMDM medium without Y-27632. HEK-293T (ATCC, Cat. CRL-1573), Vero-E6 (ATCC, Cat. CRL-1586), A549 (Cat. CCL-185), Huh 7.5 (a kind gift from Dr. Charles M. Rice, Rockefeller University), BHK-15 (a kind gift from Dr. Richard J. Kuhn, Purdue University), U-87 MG (ATCC, Cat. HTB-14), and SH-SY5Y (ATCC, Cat. CRL-2266) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, 12800-082) supplemented with 10% FBS, non-essential amino acids (NEAA, HyClone, SH30238.01), and Penicillin-Streptomycin (PS, Corning, 30-002-CI) and maintained at 37 °C with 5% CO2. C636 cells were maintained in Minimum Essential Medium (MEM, GIBCO, #41500-018) supplemented with 10% FBS and Penicillin-Streptomycin (PS, Corning, 30-002-CI) and maintained at 30 °C with 5% CO2.
ZIKV strains, cDNAs, and expression plasmids.
The prototypical African ZIKV MR-766 Uganda strain, and PRVABC-59 were obtained from BEI resources. The cDNA clone derived from the 1947 Uganda MR-766 ZIKV genome placed at the transcriptional initiation site of the cytomegalovirus (CMV) promoter49 was modified to replace Venus tag with mCherry for this study. Plasmids expressing individual ZIKV proteins and NS1 proteins from ZIKV MR-766 (pNS1-ZIKV), Dengue Virus-2 (pNS1-DENV2), West Nile Virus NY99 strain (pNS1-WNV), and Powassan virus lineage II (Deer Tick Virus) (pNS1-DTV) were generated by PCR amplification using Q5DNA Polymerase (NEB#M0492) and cloning into pCDNA3.1 or Ligation Independent Cloning into pcDNA3 mCherry LIC cloning vector (Addgene, #30125). Amino acid substitutions were introduced into ZIKV cDNA clones and expression plasmids by site-directed mutagenesis using Phusion DNA polymerase (NEB, E0553S), followed by DpnI digestion and transformation into NEB Stable Competent E. coli (New England BioLabs Inc., C3040H). Plasmids were obtained from overnight cultures of E. coli colonies grown in Luria Bertani medium using the Qiagen miniprep kit (Qiagen, 27104) or Qiagen midiprep kit (Qiagen, 12143), and sequences of the resulting clones were confirmed via Sanger sequencing at The Sequencing Core Facility at The Pennsylvania State University. DNAs were quantified using NanoDrop™ One (ThermoFisher Scientific, Waltham, MA, USA), and aliquots stored at -20 ⁰C.
To generate mutant ZIKV, cDNAs were transfected into HEK 293T cells using Lipofectamine 2000 (ThermoFisher, Cat #11668030). After 12h, the media was replaced and incubated at 37 C under 5% CO2. Cell culture supernatants were collected after 4 days of growth, filtered with a 0.45 μm mixed cellulose membrane (MCE) filter, added HEPES at a final concentration of 10 mM, and stored at -80°C. For expression of individual ZIKV proteins and flavivirus NS1 proteins, plasmids were transfected into cell lines of interest using Lipofectamine 2000. All experiments were performed under biosafety level 2 (BSL2) conditions.
ZIKA virus titration and propagation
ZIKV was propagated in African green monkey kidney (Vero) cells (ATCC, Cat. CCL-81) cultured in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, GIBCO, Cat. 11330032) supplemented with 10% FBS (Fetal Bovine Serum, GIBCO, Cat. 16140071). Confluent cells were infected with ZIKV in DMEM/F-12 2% FBS for five days, the supernatant containing viruses was harvested, and aliquots were stored at -80 ⁰C. Virus titers were determined by plaque assay of serial dilutions on Vero-E6 monolayers as described previously11,101.
Affinity purification-mass spectrometry
The affinity purification-mass spectrometry method was employed to purify His-tagged NS1 proteins using Ni-NTA resin, as previously reported56,57. We generated two plasmids expressing C terminal Octa-histidine-tagged wild type NS1 and the mutant pNS1-ZIKVΔTNT by site-directed mutagenesis. Plasmids were purified using a Qiagen Midiprep kit, sanger sequenced, and used to transfect cells grown in 150 mm dishes using Lipofectamine 2000. Briefly, the pNS1-ZIKV and pNS1ΔTNT plasmids were transfected into JEG-3 cells. At 48 hpt, cells were washed, and harvested in 1 ml PBS supplemented with Protease Inhibitor Cocktail (Millipore Sigma, P8340. The cells were lysed by sonication using a microtip attached to a sonicator (Branson), and membrane fractions were purified by ultracentrifugation at 100,000 g for 90 minutes using a TLA 120.2 rotor and Optima TLX centrifuge (Beckman Coulter). The membrane pelleted obtained after centrifugation was resuspended in 1 ml PBS supplemented with Protease Inhibitor Cocktail and 1% Fos-choline 12 and incubated at 4°C with gentle rocking for 2 h. The extracted proteins were separated from insoluble fraction by a second round of ultracentrifugation and allowed to bind to NiNTA resin for 30 min at 4°C with gentle rocking. The resin was washed with 10 column volumes of PBS with 0.01% Foscholine-12 (Anatrace, F308) followed by 2 column volumes of PBS buffer and subjected to mass spectrometry analysis at the Indiana University Proteomics Core, Indianapolis, USA. Beads were briefly resuspended in 8 M Urea, 100 mM Tris pH 8.5. Cysteines were then reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and alkylated with 10 mM chloroacetamide (CAM). Samples were diluted to less than 2 M Urea with 50 mM Tris pH 8.5 and digested overnight at 37°C with 0.5 μg Mass Spectrometry Grade Trypsin/Lys-C Mix (cat. num. V5072, Promega). Samples were filtered and acidified with formic acid (FA) before LC/MS/MS.
Nano-LC-MS/MS analysis
Nano-LC-MS/MS analyses were performed on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer coupled to an UltiMate 3000 UHPLC RSLCnano System (Thermo Fisher Scientific, Hanna-Bremen, Germany). Approximately 10 μL from each sample was loaded and concentrated using an AcclaimTM PepMapTM 100 trap column (Cat. num. 164535, Thermo Fisher Scientific) at 3 µL/min for 5 mins in 100% Buffer A (Water + 0.1% FA). Chromatographic separation was done on an Easy-Spray PepMap column (Cat. num. ES901, Thermo Fisher Scientific, ID 75 µm, 15 cm length, 3 µm particles with 100 Å pore sizes). The LC gradient consisted of holding at 3% Buffer B (Acetonitrile + 0.1% FA) for 5 minutes, followed by a gradient from 3-35% Buffer B over 75 minutes, followed by an increase to 95% Buffer B for 2 minutes, a decrease to 3% Buffer B for 2 minutes and hold at 3% Buffer B for 2 minutes.
The mass spectrometer was operated in positive mode, with a spray voltage of 1.8 kV and ion transfer capillary temperature of 250°C. Data-dependent acquisition with the top 15 most intense ions for MS/MS. Full MS scan parameters were: Resolution 70k, AGC target 3E6, m/z range 200-2000; MS2 parameters were: Resolution 17.5k; AGC target 1E5; Maximum IT 50 ms; Isolation window 4.0 m/z; Fixed first mass 100 m/z; NCE 30.0.
Mass spectrometry data analysis
The resulting RAW files from mass spectrometry experiments were analyzed using Proteome DiscoverÔ 2.5 (Thermo Fisher Scientific). The MS/MS spectra were searched against a database containing reviewed Homo sapiens proteins (downloaded from the UniProt on 10/04/2019) plus common contaminants. SEQUEST HT search engine was used with trypsin as the proteolytic enzyme, including two allowed missed cleavages, precursor mass tolerance of 10 ppm; and a fragment mass tolerance of 0.2 Da. Static modification of carbamidomethylation on cysteine residues was included, as well as dynamic or variable modifications of oxidation on methionine, peptide N-terminal methionine loss, acetylation, and methionine loss+N-terminal acetylation. Fixed PSM Validator was used as an FDR filter, and results were loaded into ScaffoldTM 4 (Proteome Software) to visualize data and calculate Fisher’s Exact test p-values with Benjamini-Hochburg correction. Relative quantitation of proteins comparing NS1 binding experiments used either Scaffold quantitation functions or Normalized Spectral Abundance Factor calculations and accounted for related isoform identifications58,102. The STRING application (Version 2.1.0)103 within Cytoscape (version 3.9.1)104 was used for visualization of functional enrichment based on protein interaction networks from observed proteins. Subcellular localization was determined from the UniProt database (Release 2023_3).
Immunofluorescence assay
Cells were grown on glass coverslips in 24 well plates and were infected with ZIKV at an MOI of 0.01. At 36 hours post-transfection, cells were fixed with 3.7% paraformaldehyde in PBS for 15 minutes and further permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature. Cells were blocked with 10 mg/ml bovine serum albumin (BSA, Sigma-Aldrich, A7906) in PBS overnight at 4°C. Subsequently, the blocking buffer was removed, and cells were treated with primary antibodies against Envelope protein (4G2, a kind gift from Theodore C. Pearson), capsid protein (GeneTex, GTX134186), or NS1 protein (GeneTex, GTX133323) followed by treatment with fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC), or Alexa Fluor 594 dye-conjugated secondary antibodies for 2 hours. Nuclei were stained using Hoechst-33342 (Invitrogen, H3570) according to the manufacturer’s instructions. Cells were washed 3 times with PBS, and coverslips were mounted onto microscope slides with FluorSave Reagent (Calbiochem, 3457) and confocal images were acquired using a Nikon A1R-MP confocal microscope fitted with a 60x oil objective lens with 1.4 numerical aperture (NA) and processed using the NIS Elements software (Nikon). Brightness and contrast were adjusted using look up tables (LUTs).
For imaging mitochondria transfer via TNTs, transfected or infected cells were co-cultured for 24 hours with cells pre-stained with 500 nM Mitotracker™ Green (Invitrogen, Cat. M7514). Samples were gently fixed in cold Phosphate-buffered saline (PBS) containing 3.5% formaldehyde methanol-free (ThermoFisher Scientific, Cat. 47392) and 0.5% glutaraldehyde (Polysciences, Cat. BLI1909) and stained with Hoechst 33342 (Invitrogen, Cat. H3570) and SiR700-Actin Kit (Cytoskeleton; CY-SC013). The specimens were prepared using mounting media (Polyscience, 18606-100) following standard protocols and imaged using the ECLIPSE Ti2 inverted microscope (Nikon).
Live-cell imaging
For screening TNT formation, cells were grown in 4-well chamber slides (IBIDI, #80427) and transfected with expression plasmids, or infected with ZIKV and ZIKV TNT- and incubated for 48 hours. Live imaging was performed in a Nikon A1R-MP confocal microscope, using a heated 60× oil immersion objective (1.4 NA) in a live imaging chamber (Tokai Hit, Fujinomiya, Shizuoka Prefecture, Japan) supplied with 5% CO2 at 37°C. Images and videos were acquired using NIS-Elements software. TNTs were determined based on z-stack image acquisition and 3D reconstruction of maximum intensity projection.
For testing virus transfer, live-cell imaging was performed as described previously with the following modifications105,106. Cells plated on a 4-chamber borosilicate cover glass (Fischer Scientific Pittsburgh, PA) at 25% confluence will be infected with fluorescent protein tagged-ZIKV or transfected with plasmids expressing fluorescently tagged proteins. The media was replaced with Opti-MEM I reduced-serum medium (Invitrogen) and stained with Hoechst 33342 stain (nucleus) and Vybrant DiD cell-labeling solution (membrane and vesicles) in conjunction with fluorescent protein-tagged viruses.
Quantification of mitochondria transfer
Transfer of mitochondria transfer via TNTs was quantified by flow cytometry. The day before, HTR-8 or JEG-3 cells were seeded in a 6-well plate at a density of 500,000 cells/well (Corning, 353046). Untransfected cells were stained 4 hours before co-culture with 150 nM MitoTracker Green FM (Invitrogen; M7514) and 8 μM CellTrace Violet (Invitrogen, C34557). Cells were washed twice with complete medium after staining and again before co-culture to remove any potential unbound dye. HTR-8 cells were transfected with 1 μg of NS1 plasmid (ZIKV, DENV, DTV) using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, L3000001). At 4 hours of post-transfection (hpt), 250,000 transfected and untransfected cells were harvested and co-cultured with homotypic or heterotypic cells at a 1:1 ratio with or without Boyden chambers (Falcon, 353090). After 24 hours post-co-culture, cells were harvested, stained with viability dye (Invitrogen, L10119), and analyzed in the BD LSRFortessa (Becton Dickinson). For THP-1 co-culture, 250,000 cells/well (Thermo Scientific, 174901) were seeded and primed with 150 nM Phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) for 24 hours and maintained in culture for three days until fully adherent and differentiated before co-culture with transfected HTR-8. Positive and negative gates were set based on FMO controls. Cells were gated for singlet, followed by live cells (Live/Dead stain) and CellTrace (Supplementary Fig. 3). The resulting cells were evaluated for NS1-mCherry and Mitotracker expression—total events = 30,000 cells.
Quantification of mitochondria accumulation
Mitochondria accumulation was quantified by flow cytometry. JEG-3 were seeded at 250,000 cells/well and cultured overnight. On the next day, uninfected cells were stained 4 hours before co-culture with 150 nM MitoTracker Green FM (Invitrogen, M7514) and 8 μM CellTrace Violet (Invitrogen, C34557). JEG-3 were either inoculated with ZIKV-mCherry (MOI=0.1) or mock infected for 2 hours and immediately co-cultured with uninfected cells at 1:1 ratio for 24 hours. For mitochondria accumulation in transfected cells, JEG-3 were either transfected with 1 μg of pNS1-ZIKV or no plasmid using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, L3000001). At 4 hpt, transfected cells were co-cultured with untransfected cells at 1:1 ratio for 24 hours. At 24 hours post-co-culture, cells were harvested, stained with viability dye (Invitrogen, L10119), and analyzed on BD LSRFortessa (Becton Dickinson). Cells were gated for singlet, followed by live (Live/Dead stain) and CellTrace (CellTrace Violet). The resulting cells were evaluated for median fluorescence intensity of MitoTracker Green to quantify accumulation of mitochondria in infected/ transfected cells. Total events = 30,000 live cells.
Interferon level analysis
JEG-3 cells were infected with ZIKV including MR-766, PRVABC-59, and ZIKVΔTNT (MOI=0.1) for 48 hours. LEGENDplex™ Human Type 1/2/3 Interferon Panel (5-plex) (BioLegend, 740350) was used to measure IFN-α2, IFN-β, IFN-γ, IFN-λ1, and IFN-λ2/3 levels simultaneously on the infected cells’ supernatant according to the manufacturer’s instructions. Bead-bound cytokines were measured using High Throughput Sampler (HTS) option on BD LSR III (Becton Dickinson) and concentrations were calculated using the LEGENDplex™ Data Analysis Software (BioLegend).
Lactate dehydrogenase (LDH) cytotoxicity assay
LDH release was measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780) according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plate at density of 1 × 104 cells/well. After overnight culture, cells were treated with Rotenone (Sigma, R8875) ranging from 0.001 to 100 μM for 24 hours. Cell culture media were collected and used for measuring LDH release. LDH levels was determined by recording absorbance at 490 nm using BioTek Epoch Microplate Spectrophotometer and the percent cytotoxicity was calculated per manufacturer’s instructions: Percent cytotoxicity = 100 × (Experimental LDH Release/ Maximum LDH Release).
Statistical analysis and reproducibility
All analyses were carried out using GraphPad Prism 9. No statistical methods were used to predetermine sample sizes. The normal distribution of continuous variables was assessed by the Shapiro-Wilk test and the statistical significance of pairwise comparisons were assessed by the Student’s t-test or the Mann-Whitney test when appropriate. Comparisons of three of more groups were performed by the one-way ANOVA followed by the Tukey’s test to determine differences between groups. Where data showed a nonparametric distribution, a Kruskal–Wallis test was used followed by post-hoc Dunn’s test. Descriptive statistics, statistical tests, and post-hoc tests for multiple comparisons are reported in each figure legends and the accompanying source data. For comparison of mitochondria transfer, five independent experiments were used. For quantification of TNTs, five randomly chosen microscope fields containing ZIKV-NS1 expressing cells from three biologically independent replicates were used for statistical analysis. For all comparisons, a two-sided P value < 0.05 was considered statistically significant.