Adenosine triphosphate (atp)

Lipopolysaccharide and adenosine triphosphate (ATP), purchased from ThermoFisher, USA, were diluted with phosphate-buffered saline (PBS) to a stock solution of 1 mg/mL and 50 µM, respectively.
MCC950 (Sigma, USA) was initially dissolved in dimethyl sulfoxide (DMSO; Calbiochem, USA) to produce a stock solution of 1000 µM followed by dilution with Roswell Park Memorial Institute Medium (RPMI 1640; Gibco, USA) serum-free media (SFM) to a concentration of 100 μM.

The medium was exchanged, and cells were incubated with DMSO (Merck; solvent of inhibitors) or MCC950 (Merck; 10 µM) for 2 h. LPS (Merck; 5 µg/mL) and viral glycoproteins (10 µg/mL) were added, and cells were incubated for another 4 h for inflammasome priming. Nigericin (Merck; 5 µM) or adenosine triphosphate (ATP) (Thermo Fisher Scientific; 5 mM) were added and incubated for 2 h for inflammasome activation. For ELISA, the supernatant was transferred to a new plate and stored at −80 °C. For cell death analysis cells were incubated another 24 h with Nigericin.

Three to five day-old mosquito females were sugar-deprived for 24 h and subsequently offered a blood meal containing a 40% volume of washed erythrocytes from SPF pig’s blood (PWG Genetics), 5% of 100 mM ATP (Thermo Scientific), 5% human serum (Sigma) and 50% volume of virus in RPMI (Gibco). The blood viral titers for both ZIKV strains were confirmed by plaque assays^{50 (link)}. Mosquitoes were exposed to the artificial blood meal for one hour using a Hemotek membrane feeder system (Discovery Workshops) with a porcine intestine membrane. Fully engorged females were selected and maintained with free access ad libitum to a 10% sugar solution in an incubation chamber with conditions similar for insect rearing until analysis.

In vitro ubiquitination assays were done as previously described (68 (link)), but modified for 48-well plates. Briefly, the ubiquitination reaction mixture contained 125 ng of E1 enzyme (Boston Biochem), 250 ng of E2 enzyme (Boston Biochem), immunopurified Cul3–KLHL15 E3 complex, 10 μg of Myc-ubiquitin (Boston Biochem), 1 μM ubiquitin aldehyde (Boston Biochem), 10 mM MgCl₂, 2 mM DTT, 10 mM creatine phosphate (Sigma), 0.5 mg/ml creatine phosphokinase (Sigma), and 20 μg of purified proteins including DCX-HaloTag-His₆ (WT or Y259L) or Halo-His₆ as control protein. The ubiquitination reactions were diluted to a final volume of 0.25 ml in 50 mM HEPES, pH 7.5, initiated upon addition of 5 mM ATP (Thermo Scientific), and incubated for up to 2 h at 37 °C on a titer plate shaker (constant speed 3, Lab-Line Instruments, Inc). Thirty-microliter reaction mixtures were sampled at the indicated times and immediately terminated in 4× Laemmli buffer. Proteins were then resolved on 8% gels by SDS-PAGE, and ubiquitinated proteins were detected by immunoblotting with Myc-Tag antibody.
Relative DCX ubiquitination in vitro was quantified by dividing Myc-Tag signals by DCX signals and GFP (KLHL15) signals in the same lane and normalizing to the zero time point (set to 1). The enzymatic kinetics was plotted in Michaelis–Menten model using GraphPad Prism.

Padlock oligonucleotides, including a 15 nt random sequence (100 nM) were ligated into circular DNA strands in the presence of template (100 nM) in 1x phi29 buffer ((Thermo Scientific), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween 20, 1 mM DTT) containing 0.01 U/μl T4 ligase (Thermo Scientific), and 1 mM ATP (Thermo Scientific). The ligation was performed at 37 °C for 30 min. To remove unligated padlock oligonucleotides, exonuclease I and exonuclease III (Thermo Scientific) were added to the ligation mix to a concentration of 0.2 U/μl and 2 U/μl, respectively. The reactions were incubated at 37 °C for 30 min and terminated by incubation at 85 °C for 20 min. Then the RCA primer (the same oligonucleotide used as ligation template) was adjusted to a concentration of 100 nM and incubated at 37 °C for 20 min to reanneal to the circular DNA strands. The RCA reactions were initiated by adding d(A, U, G, C)TP at concentrations of 1 mM and phi29 polymerase (Thermo Scientific) at 0.1 U/μl. RCA was performed at 37 °C for 10 min, and terminated by heating at 65 °C for 10 min. The RCA products were kept at −20 °C until use. The RCA products were characterized with Nanoparticle Tracking Analysis NTA (Malvern Nanosight NS300) according to the manufacturer’s instructions.

Seven day-old female mosquitoes were sugar-deprived for 24 h before infectious blood-feeding. They were offered a blood meal containing 40% of washed rabbit erythrocytes from animals housed at the IRD animal facility, 5% of 100 mM ATP (Thermo Scientific), 5% human serum (Sigma, St. Louis, MO, USA), and 50% of virus in DMEM (Gibco, Thermo Scientific). The virus titer in the blood meal was adjusted to 10⁶ FFU/mL. Mosquitoes were allowed to blood feed for one hour using a Hemotek^® membrane feeder system with a porcine intestine membrane. After blood-feeding, only fully engorged females were kept and maintained in the same insectary condition in the bio-safety level 3 facility at IRD (Vectopôle, Montpellier, France). In parallel, female mosquitoes were engorged without virus and dissected at various time points after blood-feeding under the same experimental conditions. Mosquitoes were dissected at various time points after blood-feeding (at 3, 5, 7, and 14 dpi), and the salivary gland and midgut were transferred individually to 1.5 mL Eppendorf tubes containing 350 μL of TRK lysis buffer. The blood-feeding assays were repeated three times independently with 20 to 35 dissected mosquitoes for each species and time points (R1: N = 160; R2: N = 160; R3: N = 301).

The total proteolytic activity of purified recombinant LonP1 (Abcam, ab160451) was analyzed using a Pierce Fluorescent Protease Assay Kit (Thermo Scientific^™, 23266). A mixture of 200 nM LonP1 and 10 mM MgCl₂ was prepared in BupH^™ Tris-buffered saline. LonP1 inhibitors or vehicle (DMSO) were then added, and the samples were incubated at 37°C for 1 h. Following this incubation, an equal volume of 0.04 mg/mL FITC-casein (Thermo Scientific^™, 23267), 4 mM ATP (Thermo Fisher, R0441), and 10 mM MgCl₂ was added prior to measurement using a Biotek Synergy HT plate reader. Digestion of fluorescein-labeled casein was assessed by measuring fluorescence with excitation and emission filters at 490 and 525 nm, respectively.