Cell line cultures
HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Hyclone, Logan, United States) supplemented with 10% fetal bovine serum (Sigma Aldrich, Saint Louis, United States), L-glutamine (2mM/L) and streptomycin (100 μg/ml), at 37° C in 5% CO2. MT2 cells (persistently infected with HTLV-1) and Jurkat cells infected with HTLV-1 were grown in Roswell Park Memorial Institute 1640 (RPMI) medium (Gibco, Waltham, United States) supplemented with 10% fetal bovine serum, L-glutamine (2mM/L) and streptomycin (100 μg/ml), at 37° C in 5% CO2. As for the different treatments regarding the autophagic flux, for starvation experiments, cells were incubated in Earle’s balanced salts solution (EBSS – Gibco); Spautin-1 10 μM (SP-1 – Sigma) and Chloroquine 100 μM (CQ – Sigma) were used as early and late autophagy flux inhibitors, respectively.
Infection of HeLa cells by co-culture with MT2
Infection of HeLa cells by HTLV-1 was performed by co-culture with MT2 cells. Prior to co-culture, MT2 cells were lethally irradiated at 60 Gy (6000 rads) and washed three times with 1x saline phosphate buffer (PBS 1x). HeLa cells (2x106) were cultured in a T75 flask at 30% confluence and MT2 irradiated cells (9x106) were added to the co-culture in 15 ml of DMEM with 10% fetal bovine serum, at 37 °C in 5% CO2. The co-culture was maintained for three days until HeLa cells reached confluency and reached the number of MT2 cells used (1: 1 ratio). HeLa and MT2 cells were cultured alone as controls.
Determination of the efficiency of infection by flow cytometry
The efficiency of infection of HeLa cells was determined by flow cytometry. To reveal HeLa infected cells, the viral protein Tax was labelled with Anti-Tax-Dyelight 688 antibodies (kindly provided by Dr. Tanaka of the University of Ryukyus, Japan). CD4 was labeled with anti-CD4-PercP (BD, San Jose, United States) to differentiate MT2 cells from HeLa cells. cells (1x105) were used for each condition. For intracellular labelling, cells were washed with 1 ml of PBS 1x AA (Azide/Albumin, 1000 ml of PBS 1x + 1 gr Albumin + 2 ml Azide 10%). Then the cells were treated using the commercial fixation and permeabilization solution kit (BD Cytofix/Cytoperm, San Jose, United States) according to the manufacturer's instructions. The antibodies were incubated for 40 minutes at 4°C in the dark. After incubation, the cells were resuspended in 100 μl of PBS 1x AA and analyzed by flow cytometry (BD FACS CANTO I, BD Biosciences, San Jose, United States). Data were analyzed using BD FACS Diva (BD Biosciences, San Jose, United States) and FlowJo (BD, San Jose, United States) software’s. A corresponding isotype control was used in each case.
Determination of infection by Western Blot
The Western Blot technique was performed to verify the infection of HeLa cells in co-culture where non-infected cells were used as controls. Five milliliter cultures were transferred to a pre-cooled 15 ml conical tube (infected and non-infected conditions), placed on ice and washed with 1x PBS at 4°C. After lysis with RIPA buffer, tubes were centrifuged at 14,000 rpm for 10 minutes at 4 ° C. The tubes were placed on ice and the supernatant was transferred to new tubes. Each lysate (15 μl) were taken for protein quantification (BCA protein Assay Kit) and Laemmli 4X buffer was added to the remaining material. Proteins (70 μg) were separated in 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), together with the molecular weight marker. The antibodies used included a 1:700 dilution of primary rabbit antibody anti-Tubulin (eBioscience, San Diego, United States), a 1:500 dilution of primary mouse antibody anti-Tax (Abcam, Cambridge, United Kingdom), a 1:5000 dilution of anti-rabbit-HRP (Santa Cruz, Dallas, United States) and a dilution 1:5000 anti-mouse-HRP (Santa Cruz, Dallas, United States).
Indirect immunofluorescence of Tax and LC3-II
In HeLa cells infected with HTLV-1, treatments on the autophagic flux were performed. The treatments used were starvation, CQ, starvation with CQ and SP-1. All treatments were performed at 2 h, except for SP-1 treatment that was performed at 12 h. SP-1 inhibits the activity of two ubiquitin-specific peptidases causing an increase in proteasomal degradation, which have been shown to regulate autophagy, and therefore longer incubation times are needed. Indirect immunofluorescence was performed from the treated cells. Tax and LC3-II proteins were labeled in infected and uninfected HeLa cells. Cells were fixed with chilled 4% paraformaldehyde for 10 minutes at room temperature and treated with permeabilization solution (PBS 1x + 0.05% X-100 triton) for 10 minutes. Then, the cells were incubated for 1 h at room temperature with blocking solution (PBS 1x + 10% fetal bovine serum). In each step it was washed with PBS 1x. The cells were incubated with a 1:500 dilution of primary mouse antibody anti-Tax (Abcam, Cambridge, United Kingdom) and 1:500 primary rabbit antibody anti-LC3-II (Cell Signaling, Danvers, United States) for 12 h at 4° C. It was washed three times with PBS 1x and the cells were incubated with the conjugated secondary antibodies, anti-mouse Alexa 488 and anti-rabbit Alexa 647 in a 1:700 dilution for 2 h. Finally, they were incubated with Hoechst (Thermofisher, Waltham, United States) for 15 minutes to label nucleic acids and mounted with polyvinyl acetate (PVA). This protocol was also used to observe the distribution of Tax and LC3-II in HTLV-1 infected Jurkat cells and persistently HTLV-1 infected MT2 cells. Unlabeled cells were used as control to establish autofluorescence in each case. Images were acquired with a Carl Zeiss LSM 800 spectral confocal microscope. The processing, analysis and quantification of the images were carried out with the FIJI software.
Relative quantification of HTLV-1 mRNA expression by qPCR from treatments on autophagic flux
In the MT2 cell line, treatments on the autophagic flux were performed to quantify the relative expression of HTLV-1 mRNA. The treatments used were starvation, chloroquine, starvation with chloroquine and SP-1. All treatments were performed at 1 h, 2 h and 3 h, except for SP-1 treatment that was performed at 12 h. Total RNA was isolated by using TRIzol (Invitrogen). Total samples were treated with RQ1 RNase-Free DNase (Promega) and 1 μg of total RNA were reverse-transcribed into complementary DNA by using a MMLV (Promega) according to the manufacturer's instructions. Pol, tax and p19 mRNA expression was measured by qPCR with Eva green reagents (Solis BioDyne) and GAPDH gene expression was used as an internal control. The expression of the viral genes was normalized to GAPDH by using the (2-∆∆Ct) ratio.
Mean fluorescent intensity (MFI) of Tax after treatments on autophagic flux
In the MT2 cell line, expression levels of Tax viral protein were assayed in function of treatments on the autophagic flux. Cells were subjected to starvation, chloroquine and the combination of both for 2 h. For intracellular labelling, cells were washed with 1 ml of PBS 1x AA (Azide/Albumine, 1000 ml of PBS 1x + 1g Albumin + 2 ml Azide 10%). Then the cells were treated using the commercial fixation and permeabilization solution kit (BD Cytofix/Cytoperm, San Jose, United States) according to the manufacturer's instructions. The cells were incubated with a 1:500 dilution of primary mouse antibody anti-Tax (Abcam, Cambridge, United Kingdom) for 5 h at 4°C in darkness. Then it was incubated with a 1:700 the secondary antibody anti-mouse (Alexa 488) for 2 h at 4°C in the darkness. After incubation, the cells were resuspended in 100 μl PBS AA 1x and analyzed by flow cytometry (BD FACS CANTO I, BD Biosciences, San Jose, United States). The results were analyzed using BD FACS Diva (BD Biosciences, San Jose, United States) and FlowJo (BD, San Jose, United States) software. The corresponding isotype control was used in each case.
Statistics
To study the significance between groups, a one-way ANOVA was used. Values were expressed as mean ± SEM. All significance tests were 2-tailed, and the level of significance was set at 0.05. All cell lines data shown are representative of at least 5 independent experiments. Statistical tests were performed with the SPSS 21 program.