Zika virus triggers autophagy to exploit host lipid metabolism and drive viral replication

Isolation, culture, and titration of ZIKV

Vero and Vero E6 cells were seeded at 3 × 106 cells in 75 T flasks and allowed to attach overnight. Cells were infected with ZIKV-MR766 (ATCC® VR-84™) at a MOI (Multiplicity of Infection) of 0.1 for 2 h (h); then cells were covered with DMEM with 2% FBS. After three days at 37 °C and a humidified atmosphere, the supernatant was collected, and cell debris was separated by centrifugation at 2000 rpm at 4 °C for 10 min. The supernatant, containing mature virions, was collected, aliquoted, and stored at – 80 °C.

The viral titer was then determined by the traditional plaque assay as follows: Vero E6 cells were suspended and approximately 2.5 × 105 cells were allowed to attach overnight in 12 well plates, in DMEM supplemented with 10% FBS and 1% penicillin. The following day, confluent monolayers were infected with tenfold serial dilutions of virus suspension and the virus was permitted to attach for 2 h at 37 °C. Infected cells were rinsed once with 1X PBS and then covered with the agar overlay, containing 50% low melting point agar (# V2111, Promega), 40% 2X DMEM and 10% FBS. The agar overlay was allowed to solidify at room temperature (RT), and the cells were incubated for five days at 37 °C to facilitate plaque development. Before plaque count, cells were fixed with 4% formaldehyde (# F8775, Sigma) for 20 min. The solidified agar was removed, and cells were washed with 1X Phosphate Buffered Saline (PBS) and stained with a 1% crystal violet solution (# C0775, Sigma) for 10 min. Plaques were counted, and the virus titer was expressed as PFU/ml.

Cell culture and treatments

MDCK (Madin-Darby Canine Kidney Epithelial Cells, ATCC®-CCL-34™), Vero (African green monkey kidney epithelial cells, ATCC® CCL-81™), and Vero E6 cells (ATCC© CRL-1586™) were purchased from the American Type Culture Collection (ATCC). MDCK, Vero and Vero E6 cells were grown in Dulbecco's modified Eagle medium (DMEM, catalog # 12800017, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher, # 16000044) and 1% penicillin–streptomycin (PS, Invitrogen, # 15140122). All cell lines were incubated at 37 °C under a humidified atmosphere consisting of 5% CO2 and 95% air.

As described in Ghosh Roy et al. [20], cells were seeded at 2.5 × 105 cells per well in a 6-well plate and allowed to attach overnight. The next day, ZIKV MR766 (ATCC® VR-1838TM) was added to the cells at an MOI of 1, and the cells were incubated for 2 h before washing with and adding fresh media. The infected cells were further cultured for 12, 24, or 48 h depending on what we were testing. However, most of the data presented represent experiments terminating at 48 h. We also examined the role of the PERK pathway by exposing cells to class I/III PI3K inhibitor Wortmannin (wort, #681675, Calbiochem) at 50 μM, HMG-CoA reductase inhibitor Atorvastatin (ATV, #Y0001327, Sigma) at 5 μM, Salubrinal (Sal, #sc-202332, Santa Cruz) at 3 μM, or Tunicamycin (Tunica, #T7765, Sigma) at 3 μM. In all these cases, cells were incubated with inhibitors for 1 h prior to infection.

For certain experiments cells were treated with 50 nM bafilomycin A1, a vacuolar H+-ATPase (V-ATPase) inhibitor used to inhibit autophagy, either before or after infection. For bafilomycin pretreatment, cells were seeded as described above and treated the next day with 50 nM bafilomycin. After one hour the medium was aspirated and the cells were incubated with ZIKV at MOI 1 for 2 h. After the incubation period was over, fresh cell culture media was added to the cells and allowed to grow for 12, 24 or 48 h.

Immunofluorescence

Immunofluorescence was performed as described in Lin et al. [21]. MDCK were grown on glass coverslips to 70% confluence. Appropriate treatments were done as described above. At the indicated endpoint of each treatment, cells were washed twice with 1X PBS, and fixed with 4% paraformaldehyde for 1 h at RT. Cells were permeabilized with 0.2% Triton X-100 (#X100, Sigma) in 1X PBS for 15 min at 37 °C. Then, cells were washed three times with 1X PBS for 5 min each. Cells were treated with 1% BSA (# A2153, Sigma) 0.1% Triton X-100 in 1X PBS for 1 h before addition of antibodies. Cells were incubated overnight at 4 °C with various antibodies depending on the experimental needs. To check for the presence of Zika infection a Anti Zika E-protein rabbit polyclonal Ab was used at a dilution of 1:200 (# Ab00230-23.0, Absolute antibody) or a viral E protein mouse monoclonal antibody (isolated from Hybridoma cells, ATCC® HB-112) with 1:10 dilution. To stain for LC3 puncta Anti-LC3B polyclonal rabbit Ab was used at a dilution of 1:100 (#L7543, Sigma). Other antibodies used and the respective concentrations can be found in Additional file 1: Table. Following overnight incubation, cells were washed three times with 1X PBS for 5 min. Cells were incubated with appropriate secondary antibodies for 1 h at room temperature at a dilution of 1:1000 (see Additional file 1: Table). They were then washed with 1X PBS for 5 min and then stained with 4, 6 -diamidino-2-phenylindole (DAPI) (1 mM) (# ab228549, ABCAM) for 8 min. Cells were washed twice with 1 × PBS, mounted, and embedded in Fluoromount® and observed at 40X and 100X by fluorescence microscopy using the Leica Leitz DMRB microscope. Details of all antibodies are described in Additional file 1: Fig. 6S.

Filipin staining

Filipin III was dissolved in anhydrous dimethylformamide under inert gas conditions, to form a stock concentration of 1 mg/ml. Filipin was diluted 1:20 to 0.05 mg/ml in PBS and added to cells with the secondary antibodies on day 2 of immunofluorescence in complete darkness for two hours.

Lipid droplet staining and quantification

For measurement of lipid generation, cytological analysis of LD using Oil Red O (ORO) (# O0625, Sigma), a fat-soluble dye that stains lipids, was performed as described previously [22].

Briefly, after treatment and fixation, cells on coverslips were washed with 60% isopropyl alcohol and then dried for a few hours or overnight. Staining can be performed alone or during immunofluorescence after the secondary antibody incubation. LD in samples were then stained with 60% ORO solution for 20 min and coverslips were rinsed four times with distilled water. Samples were then mounted on glass slides with Fluoromount® and visualized with the same fluorescence microscope. The total red fluorescence per cell was quantified using ImageJ software. (ImageJ, along with its updated version Fiji, is a free yet powerful image analysis/statistics package developed by personnel of the National Institutes of Health (USA), downloadable at ImageJ (nih.gov). The numbers were reported calculated by multiplying the mean fluorescence of the cell by the area of the cell. This was done for every cell in each frame. Their averages were reported as mean total ORO/cell in arbitrary units. At least 200 cells from different sections of a given slide were used in our quantification. Each experiment was done at least 3 times. Examples of how the quantification was done are found in Additional file 1: Fig. 5S.

Quantitative RT-PCR

Cell lines were infected and treated as described above. According to the manufacturer’s protocol, total mRNA was isolated from mock-infected and ZIKV-infected cells with the GenElute™ Total RNA Purification Kit (Sigma, # RNB100). Power SYBR™ Green RNA-to-Ct™ 1-Step Kit (catalog no. 4391178, Thermo Fisher) was then used to reverse-transcribe and obtain the cDNA, followed by real-time PCR. The following primers were used to quantify the target gene (ZIKV-NS1) and the loading control (beta-tubulin):

NS1 forward primer 5′ TACACCCAGTCACAATAGGAGAGTG 3′/reverse primer 5′ CCATGCATTCATTGTCACACTTGTGG 3′, tubulin forward primer 5′ AGGATTCGCAAGCTGGCTG 3′/reverse primer 5′ TAATCCACAGAGAGCCGCTCC 3′.

Statistical analysis

The total fluorescence for LD reported was analyzed using ImageJ as reported previously [22]. Briefly, a random field of cells was selected. Each cell within the field was circled and analyzed for the area of the cell and the intensity of the red channel (LD channel). The product of these values gave a quantity for each cell which was averaged for each condition and compared between experimental conditions. Their averages were reported as mean total ORO/cell in arbitrary units.

Statistical significance of the results was calculated by two sample unequal variance t test using a two-tail distribution through excel; values of P < 0.05 represent no statistical difference between compared samples. Pearson Correlation Coefficient was calculated using excel. The average of each time point (12 h, 24 h, 48 h) was taken for Normalized Ct values and Average ORO as a measure of lipid quantification, and these values were used to generate the Pearson Correlation Coefficient in Additional file 1: Fig. 1SC.

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