Cell lines and viruses

Human Embryonic Kidney (HEK)-293T cells (ATCC CRL-1573) were grown in Dulbecco’s Modified Essential Medium (DMEM) (Thermo Scientific) supplemented with 10% fetal bovine serum (FBS) (Sigma). HEK-293T cells stably transfected with a firefly luciferase reporter gene driven by the IFN-β promoter (293T–IFN-β–FFLuc), previously described in [26], were grown as stated for HEK-293T cells. BHK-21 (ATCC CRL-6281 C) and Vero (ATCC CCL-81) were grown in DMEM supplemented with 5% FBS. Peripheral blood monocyte (THP-1) cells (ATCC TIB-202) were grown in Roswell Park Memorial Institute (RPMI) medium (Thermo Scientific) supplemented with 10% FBS. Sheep thymus (ST) cells, an established cell line produced from primary thymus cells [27] were grown in DMEM supplemented with 10% FBS. All cells were kept at 37 °C in the presence of 5% CO2. Bluetongue virus serotype 8 (BTV-8, Belgium/06) was used in all the experiments. BTV stocks were prepared by infecting BHK-21 cells with a MOI of 0.1. Supernatants were collected at 48 h post-infection (hpi), and after freeze/thaw cycles and sonication, the supernatants were clarified and stored at -80ºC until use. Reverse genetic BTV-8 with a deficient NS3 encoding ORF (BTV-8 ΔNS3) and its control (rg BTV-8) were prepared as described by [28]. Modified Vaccinia Ankara virus (MVA) was kindly provided by Dr. Javier Ortego (CISA-INIA/CSIC).

Virus inactivation

BTV-8 was inactivated with 1 mM binary ethyleneimine (BEI) at 37ºC for 48 h. Briefly, 1mM BEI was prepared by dissolving 2-bromoethylamine hydrobromide (BEA) in 0.7% sodium hydroxide (NaOH). The solution was incubated at 37ºC for 1 h to allow BEA to get converted to BEI, which was then sterilized using 0.2 μm filters. 3% BEI was added to virus suspension and the inactivation was carried out at 37ºC for 48 h with an intermediate change of flask at an 18-hour interval.

Plasmids

Plasmids encoding BTV NS3 and NS4 open reading frame (ORF) were cloned into pIRES-cOFP-COOH-FLAG (Promega) expression vector. Plasmid encoding BTV NS3 protein (NS3-FLAG-pIRES-cOFP) was used as template to generate NS3 non-ubiquitinated mutant by substituting K amino acid residues 13 and 15 (NS3- K13,15), as described in [24]. pLenti-CMV-cGAS-HA plasmid, encoding human cGAS, was a gift from Jonathan Kagan (Addgene plasmid #130910; http://n2t.net/addgene:130910; RRID: Addgene_130910). pcDNA3.0-STING-FLAG plasmid, encoding human STING, was kindly provided by Dr. Subhash G Vasudevan (Duke-NUS Graduate Medical School, Singapore). pCAGGs-Denv-NS5-HA plasmid, encoding dengue virus NS5 protein was provided by Dr. García-Sastre (Icahn School of Medicine at Mount Sinai, New York, USA).

Interferon production assays and quantitative PCR

ST cells were treated with mock or BEI inactivated BTV (iBTV) or infected with BTV-8 at a MOI of 5.0 (or its equivalent prior to inactivation for iBTV). Six hours after primary treatment, cells were either treated with mock, infected with MVA at an MOI of 2.0, or transfected with E.coli DNA (1 µg / 2.0 × 105 cells) using TransIT®-LT1 (Mirus Bio). Twelve hours post-secondary treatment, nucleic acids were isolated using RNeasy Micro Kit (Qiagen) according to the manufacturer´s guidelines. Quantification of ovine Ifnα and Isg15 transcripts was determined by normalizing gene expression to β-actin gene expression, and relative expression levels were calculated using the 2−ΔΔCt method [29], where ∆Ct = Ct gene of interest (Ifnα or Isg15) – Ct housekeeping gene (β-actin) and ∆∆Ct = Sample ∆Ct average - Control group (mock) ∆Ct average). Quantitative real-time PCR (qPCR) and reverse transcription (RT)-qPCR were carried out to verify MVA and BTV infection respectively, using Luna Universal One-Step (New England Biolabs). For this purpose, cells were collected and pelleted to remove supernatant and washed twice with PBS (phosphate buffered saline) before nucleic acids isolation.

Transcription levels of Ifnα, Isg15 and β-actin genes were evaluated by RT-qPCR using SYBR Green I Master Reagents (Roche). All PCRs were performed on a Light Cycler 480 System instrument (Roche). Primer sequences are available upon request.

Interferon-ß reporter assay

HEK-293T cells stably expressing firefly luciferase under the control of an IFN-ß promotor (293T–IFN-β–FFLuc) were used to study the ability of BTV NS3 protein to inhibit the induction of the IFN-ß reporter. Briefly, 50,000 cells were transfected using TransIT®-LT1 (Mirus Bio) with 100ng total DNA composed of a mix of the following plasmids: pcDNA3.1-empty, pLenti-CMV-cGAS-HA, pcDNA3.1-STING, pIRES-BTV-NS3-FLAG or pIRES-BTV-K13,15/R-NS3-FLAG (K13,15/R-NS3 non-ubiquitinated mutant). Twenty-four hours post-transfection, IFN-ß promotor induction was measured using the neolite luminescence reporter gene assay system (PerkinElmer) as per the manufacturer’s protocol.

Western blots

HEK-293T or THP-1 cells were mock treated or infected with BTV-8 (MOIs 0.1, 1.0 and 5.0) or transfected with our experimental control and control plasmids using TransIT®-LT1 (Mirus Bio), following the manufacturer´s protocol. Twenty-four hours later, cells were washed with PBS and lysed on ice for 30 min with RIPA lysis buffer (Sigma) supplemented with complete protease inhibitor (Roche). Lysates were centrifuged for 15 min at 20,000 xg at 4ºC to remove cell debris. Cell lysates were re-suspended, in 2X Laemmli sample buffer (Bio-Rad) supplemented with 2-mercaptoethanol and boiled at 100 °C for 10 min. The samples were loaded in polyacrylamide–SDS gels and the proteins were electrophoretically separated. Proteins were then transferred to PVDF membranes. Blots were blocked with tris-buffered saline (TBS) with 0,05% Tween 20 (Thermo Scientific) and 5% milk for one hour at room temperature (RT). Blots where then incubated with the following primary antibodies: anti-cGAS (D1D3G), anti-STING (D2P2F), anti-ATG7 (D12B11), anti-LC3A/B (D3U4C) (all from Cell Signaling Technology), anti-FLAG (F7425), anti-ß-actin (A2228) and anti-haemagglutinin (HA) (H3663) (all from Sigma Aldrich). Secondary HRP-conjugated anti-mouse or anti-rabbit IgG (GE Healthcare) antibodies were used, and protein bands were visualized by chemiluminescence (ECL Plus, Thermo Scientific). Immunoblot image acquisition was performed on a ChemiDoc Imaging System (Biorad). Densitometry analysis of cGAS, STING and β-actin expression was performed using ImageJ software.

Proteasome and autophagy pathway inhibition

HEK-293T cells were transfected with plasmids encoding cGAS and BTV-NS3 or an empty vector. Twenty-four hours post-transfection, cells were treated with proteasome inhibitors MG132 (0.2, 2 or 20 µM) and lactacystin (20 µM), autophagosome formation inhibitor 3-MA (5 mM) (all from Sigma), or autophagosome maturation inhibitor, bafilomycin A1 (BAF-A1, 1, 10 or 100 nM) (InvivoGen). Six hours post-treatment, cells were lysed, and proteins were analyzed via Western blot. For ATG7 gene silencing, HEK-293T cells were transfected with 100 nM of ATG7 siRNA or control siRNA (Cell Signaling Technology) for 48 h and transfected with plasmids encoding cGAS and BTV-NS3 for an additional 24 h. All transfections were carried out using jetPRIME® transfection reagent (Polyplus). Cell lysates were collected and analyzed by SDS-PAGE and immunoblotting.

cGAS inhibition assay

THP-1 cells were mock treated of infected with BTV-8 (MOI 0.1) and 1 h after infection cells were treated with cGAS inhibitor G140 (20 µM) (InvivoGen). At 16 h post-infection, cells were harvested for determination of viral replication by plaque assay and qPCR.

Immunoprecipitation assays

HEK-293T cells were co-transfected with plasmids encoding HA-tagged cGAS and FLAG-tagged BTV-NS3. Twenty-four hours post-transfection, cell lysates were collected as described in the Western blot section. A portion of the lysates was separated and kept as whole cell extract (WCE), while the other portion was used to perform the immunoprecipitation (IP) assay. Lysates were incubated with anti-HA or anti-FLAG affinity gel antibody beads (Sigma) overnight at 4ºC under rolling agitation. After incubation, the affinity gel samples were washed four times for 10 min in lysis buffer. The affinity gel samples were then re-suspended in 2X Laemmli buffer with 2-mercaptoethanol and boiled at 100ºC for 10 min. SDS–PAGE followed by immunoblot analysis was performed on the IP and WCE.

Immunofluorescence

Vero cells were seeded on coverslips (Thermo Scientific) in 12-well plates and then mock treated or infected with BTV-8 at an MOI of 5.0. Twenty-four hpi, cells were washed three times with PBS before fixing and permeabilizing with 100% ice cold Methanol (Thermo Scientific) for 10 min at -20ºC. Nonspecific binding sites were blocked with Dako antibody diluent (Agilent) for 1 h at RT. Cells were incubated with primary antibodies overnight at 4ºC, after which they were washed three times with PBS and incubated for 1 h at RT with secondary antibodies. After washing, nuclei were stained with 10 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) and coverslips were mounted using Prolong Gold antifade reagent (Invitrogen). The following antibodies were used: primary antibodies anti-VP2-BTV8 rabbit serum produced in our laboratory [30], anti-ssDNA mouse (MAB3034: EMD Millipore), anti-TOM20 mouse (SC17764, Santa Cruz), and secondary antibodies anti-mouse IgG conjugated to Alexa Fluor 647 and anti-rabbit IgG conjugated to Alexa Fluor 488 (Invitrogen).

Confocal microscopy image acquisition

Images were captured using a LSM 880 confocal microscope (Zeiss) fitted with a Plan Apochromatic ×63/1.4 or ×40/1.4 oil objective. Images were collected at a resolution of 1,024 × 1,024 pixels. Image processing was carried out using Fiji/Image J software.

Mitochondrial membrane potential assay

JC-1 - Mitochondrial Membrane Potential Assay Kit (Abcam) was used to evaluate the ability of BTV to modify mitochondrial membrane potential. Tetraethylbenzimidazolylcarbocyanine iodide (JC-1) is a cationic dye that accumulates in energized mitochondria. At low concentrations (due to low mitochondrial membrane potential), JC-1 is predominantly a monomer that yields green fluorescence with emission of 530 ± 15 nm.

At high concentrations (due to high mitochondrial membrane potential), the dye aggregates yielding a red to orange colored emission (590 ± 17.5 nm). Therefore, a decrease in the aggregate fluorescent count is indicative of depolarization whereas an increase is indicative of hyperpolarization. Following the manufacturer´s guidelines, ST cells were seeded in 24-well plates and mock-treated or infected with BTV at MOIs of 0.1, 1.0 and 5.0. For iBTV treatment, cells were incubated with the equivalent to a MOI of 5 prior to inactivation. Twenty-four hpi, cells were treated with 25 µM tetraethylbenzimidazolylcarbocyanine iodide (JC-1) for 10 min at 37ºC, after which fluorescence was analyzed by flow cytometry. As a depolarization control, cells were treated with 100 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) for 1 h at 37ºC prior to JC-1 addition. Samples were acquired on a BD FACSCelestaSorp flow cytometer and analyzed using the FlowJo software (Tree Star Inc, USA).

Intracellular BTV-VP7 staining for detection of cell infection

Intracellular staining for BTV VP7 protein was carried out to confirm BTV infection of ST or HEK-293T cells in mitochondrial membrane potential assay and cGAS degradation assay, respectively. Twenty-four hours post-infection, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ (BD Biosciences) for 20 min on ice. After two washes with 1×BD Perm/Wash™ buffer (BD Biosciences), cells were stained with anti-BTV-VP7-FITC-conjugate mouse antibody (VMRD) for 30 min on ice. After two more washes with PBS, cells were analyzed by flow cytometry.

Statistical analysis

Statistical analysis was carried out using Prism 8.0 software (GraphPad Software Inc). Data were analyzed with one-way ANOVA with Tukey´s multiple comparisons test. P-values were considered statistically significant when p < 0.05.