Leupeptin

Cell lysates were produced as described above. 2 μl of the cell lysates were incubated with 20 μM fluorogenic CTSB substrate (Z-RR-AMC; Enzo; #BML-P137-0010) diluted in lysis buffer (50 mM sodium acetate, 0.1 M NaCl, 1 mM EDTA, 0.2% Triton X-100, pH 4.5) at 37°C for 30 min. Recombinant CTSB [produced in-house, as recently described for CTSD (Marques et al., 2019 (link))] and the addition of the CTSB inhibitor Leupeptin (Enzo; #ALX-260-009-M025), were used as positive and negative controls. Detection was performed with an Infinite 200 PRO M Plex multimode microplate reader (ex.: 360 nm; em.: 440 nm; Tecan Trading AG; #TEC006418I).

Antibodies against CD8 and rabbit HA were from Sigma, antibodies against BAP31, LAMP1, actin, Hsp70 and β-tubulin were from AbCam, anti-ERGIC53 antibody was from Alexis, mouse anti-HA antibody was from Santa Cruz Biotechnology, anti-BiP antibody was from Cell Signaling, anti-CNX and -CRT antibodies for immunoblotting were from Stressgen, anti-CRT antibody for immunofluorescence was from Thermo Scientific and anti-GM130 antibody was from BD Biosciences. Antibodies against opsin and STT3B antibodies were provided by Stephen High (University of Manchester, Manchester, UK). IRDye 800 CW and IRDye 680 RD were from LI-COR, and secondary antibodies for microscopy were from Jackson Laboratories (Stratech Scientific). The inhibitors leupeptin (Enzo Life Sciences), pepstatin A (Sigma), Z-LLF-CHO (PSII, Calbiochem), chloroquine (Sigma) and cycloheximide (CHX, Sigma) were used at 0.5 mM, 1 µg/ml, 10 µM, 5 mM and 100 µg/ml, respectively.

Cells were incubated with 10 μM proteasomal inhibitor II (PSII, (benzyloxycarbonyl)-Leu-Leu-phenylalaninal) (Merck Millipore, Nottingham, UK) or a combination of 100 μM leupeptin (Enzo Life Sciences, Exeter, UK) and 1 µg ml⁻¹ of pepstatin A (Sigma) for 5-6 h. When 25 nM Bortezomib (Velcade) (Selleck Chemicals, Stratech, Newmarket, UK) was used the incubation time was 16-24 h. Small molecules [2.5 mM 4PBA (Sigma), 50 mM TMAO (Sigma), 1 mM TUDCA (Calbiochem, Merck Millipore), 50 nM 17-AAG (Sigma), 50 nM celastrol (Sigma)] were added to the cultured media 24 h prior to harvesting, fixation or patch clamp analysis.

DCs or Møs (1x10⁶ cells/mL) were exposed to the following protease inhibitors for 45 minutes: 10μM MG132, 5μM E64, 10μM cathepsin S inhibitor Z-FL-COCHO, 10μM leupeptin (Enzo Life Sciences), 120μM bestatin (Sigma-Aldrich), before incubation with different concentrations of recombinant HIV-1 p24-protein for 1hr at 37°C or 4°C. Samples were thoroughly washed in ice-cold PBS and immediately treated with 3mg/mL pronase E (Sigma-Aldrich) in AIM-V media without serum for 10 minutes on ice. Cells were washed, lysed in 0.5% Triton X-100 containing lysis buffer and the amount of p24 protein in cell lysates was measured using a standard HIV-1 p24 antigen ELISA (Perkin Elmer).

To measure CTSD activity, 5 µg of cell lysate freshly enriched for lysosomes or mouse brain lysate was incubated in 100 µl lysis buffer (50 mM sodium acetate, 0.1 M NaCl, 1 mM EDTA, 0.2% Triton X-100) containing 10 µM quenched fluorogenic peptide (Enzo, New York, NY; #BML-P145) and 25 µM leupeptin (Enzo, #ALX-260-009-M025) at 37 °C for 30 min. The addition of CTSD inhibitor pepstatin A (PepA; Sigma-Aldrich, St. Louis, MO; #P5318) was used as a negative control. Fluorescence signal was measured for each sample in triplicates with a plate reader (SpectraMax Gemini, Molecular Devices, San José, CA, excitation: 322 nm; emission: 381 nm). The determination of CTSB and CTSL enzymatic activities was done under the same conditions, utilizing 20 µM quenched fluorogenic peptide (Enzo, #BML-P139-0010) for CTSB and 9.4 µM (BioRad; #ICT942) of fluorescent probe for CTSL. Fluorescence signals were measured at excitation: 365 nm and emission: 440 nm for CTSB, and excitation: 590 nm and emission: 628 nm for CTSL. All values were corrected for background fluorescence.

According to the results of proteolysis assays as above, the complex of 500 nM rHslU and 100 nM rHslV (rHslU₅rHslV₁) were pre-treated at 37 °C for 30 min with 20 μM MG132 (ENZO Life Sciences), an inhibitor of threonine, serine and cysteine proteases/peptidases, 5 μM 4-hydroxy-5-iodo-3-nitrophenylacetyl-leu-leu-leu-vinylsulfone (Calbiochem, Darmstadt, Germany) and 100 μM lactacystin (ENZO Life Sciences), the inhibitors of threonine proteases/peptidases, 20 μM leupeptin (ENZO Life Sciences), an inhibitor of serine and cysteine proteases/peptidases, and 1 mM E64 (ENZO Life Sciences), an inhibitor of cysteine proteases/peptidases, respectively.^{22 (link), 33 (link)} The subsequent experimental steps to detect the hydrolysis of FITC-casein, Suc-LLVY-AMC, Suc-AAF-AMC and Z-GGL-AMC substrates were the same as described as above.

Cells were seeded in 6-well plates 24 h prior to any treatment with the following compounds: bafilomycin A1 (BAF.A1, LC Laboratories, Woburn, MA, USA, R-5000, B-1080, 100 nM) [18 (link)]; rapamycin (RAPA, Fisher, Pittsburgh, PA, USA, BP2963.1, 1 µΜ) [19 (link)], Hegf (E9644, 100 ng/Ml), pepstatin A (P5318, 50–100 µM), DMSO as a vehicle (dilution factor 1:1000), all from Sigma-Aldrich; and leupeptin (Enzo, ALX-260–009, Farmingdale, NY, USA, 50–100 µM).