D luciferin

Twelve hours after transfection with Mitochondrial-targeted luciferase enzyme (mtLUC), cells were re-plated into a 96-multiwell White/Clear Bottom Plate, TC Surface (Thermo Fisher) (10x10³ cells/well) for the luminescence measurements by a PerkinElmer EnVision plate reader equipped with two injector units. For the ATP measurements, cells were washed twice with a modified Krebs Ringer Buffer (KRB: 135mM NaCl (Sigma-Aldrich), 5mM KCl (Sigma-Aldrich), 0.4mM KH2PO4 (Sigma-Aldrich), 1mM MgSO4(7 H2O) (Sigma-Aldrich), 1mM MgCl2 (Sigma-Aldrich), 20mM HEPES (Sigma-Aldrich), pH 7.4), glucose (0.1% D-(+)-Glucose (Sigma-Aldrich))/Ca²⁺ (1mM Calcium chloride solution (Sigma-Aldrich) and placed in 50 μL of KRB + glucose/Ca²⁺. After recording the basal background signal for 5 s (s), a 20 μM (final concentration) D-luciferin (Duchefa Biochemie) solution in KRB + glucose/Ca²⁺ was added and, since luciferin is cell-permeable, the luminescence was detected immediately. After 100 s, when the signal reached a plateau, a 100 μM (final concentration) histamine solution in KRB + glucose/Ca²⁺ with 20 μM (final concentration) D-luciferin was added to boost ATP production. For each measurement, the luminescence signal (CPS, counts per second) after histamine was normalized on the mean CPS at the plateau reached after the first luciferin addition.

For LUC activity measurements in stable transformed Arabidopsis thaliana plants were sprayed with 1 mM D-luciferin (Duchefa, Haarlem, NL) 24 h and 1 h before imaging with an (− 80 °C) air-cooled CCD Pixis 1024B camera system (Princeton Instruments, Massachusetts, USA) equipped with a 35 mm, 1:1.4 Nikkon SLR camera lens (Nikon, Tokyo, Japan) fitted with a DT Green filter ring (Image Optics Components Ltd, Orsay, France) to block chlorophyll fluorescence. Exposure time is as indicated.
For transient assays, N.benthamiana leaves were harvested 4 days post agro-infiltration. Leaves were sprayed with 1 mM D-luciferin at 24 and 1 h before imaging (5 min exposure time). Relative luminescence from LUC activity was analysed in Image J (Bethesda, Maryland, USA), using background subtraction. For each treatment LUC activity in leaves from 6 to 8 independent plants was quantified.

The TZA assay (33 (link)) was slightly modified to measure virus production from PWH PBMCs. PBMCs from the HIVRRA assay were co-cultured with TZM-BL cells in a 96-well plate for 4 days. LTR-driven luciferase activity from TZM-BL cells co-cultured with PBMCs from PWH was measured by addition of 25 µL of luciferase activity reagent (LAR) substrate (0.83 mM ATP, 0.83 mM of d-Luciferin [Duchefa Biochemie B.V., Haarlem, the Netherlands], 18.7 mM MgCl₂, 0.78 µM Na2H2P2O7, 38.9 mM Tris [pH 7.8], 0.39% glycerol, 0.03% Triton X-100, and 2.6 µM dithiothreitol) and measuring luminescence in relative light units (RLUs) using a luminometer (Berthold Technologies, Germany). The relative infectious units per million (IUPM) cells were calculated at 30% of the maximum RLU using logistic regression.

To analyze the effect of SERINC3, SERINC5 and the primary Nef proteins on HIV-1 replication, U87 CD4+CCR5+ cells were inoculated with NL4-3 luciferase Bal26-pseudotyped single-round reporter viruses produced in the presence or absence of Nef alone or in combination with SERINC3 or SERINC5. Equal amounts of reporter viruses, as determined by an in-house p24 ELISA, were used to inoculate the target cells [23 (link)]. Luciferase activity was determined, as a measure of viral replication, 48 h post-infection by adding 25 µL substrate (0.83 mM ATP, 0.83 mM d-luciferin (Duchefa, Haarlem, The Netherlands), 18.7 mM MgCl₂, 0.78 µM Na₂H₂P₂O₇, 38.9 mM Tris pH 7.8, 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100 and 2.6 µM dithiothreitol) directly to the culture medium. Luminescence was measured for 1 s per well using a luminometer (Berthold, Bad Wildbad, Germany). The infectivity of the virus produced in the presence of the primary Nef and SERINC3/5 was corrected for the infectivity of the virus produced in the presence of the same primary Nef and the relevant empty vector. The ability of the primary Nef proteins to counteract SERINC3/5 was expressed relative to NL4-3 Nef (fold change).

Donor PBMC were stimulated for 3 days with Phytohemagglutinin (PHA; 1 μg/ml) in Iscove's modified Dulbecco medium (IMDM) supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 U/ml) and ciproxin (5 ug/ml) at 4 × 10⁶/ml cells. Stimulated PBMC were then plated at 3 × 10⁶ cells per well in IMDM supplemented with IL-2, 10% FCS, penicillin, streptomycin and ciproxin and infected with HIV-1 NL4.3-BaL. At day 7, cell pellets and viruses were harvested. Tissue culture infectious doses (TCID₅₀) of the produced viruses were determined on TZM-bl (4000 cells/well). Luminescence was determined after 3 days using an in-house luciferase assay: 25 μl of luciferase substrate (0.83 mmol/l ATP, 0.83 mmol/l dluciferin (Duchefa Biochemie B.V., Haarlem, The Netherlands), 18.7 mmol/l MgCl₂, 0.78 μmol/l Na₂H₂P2O₇, 38.9 mmol/l Tris (pH 7.8), 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 μmol/l dithiothreitol) was added per well. Luminescence was measured using a luminometer (Berthold Technologies, Bad Wildbad, Germany) and is expressed as Relative light units (RLU).

To obtain Jurkat T cells with a stable ADAR1 knockdown, the cells were inoculated with either the LV-shRNA NT-control or the LV-shRNA targeting ADAR1 at a MOI of 10. Cells were subsequently cultured in the presence of puromycin (2 μg/ml) for several passages to obtain a stable transduced cell line. Jurkat T cell lines and IL-2 stimulated PBMCs were infected with YU2, YU2-GFP or the VSV-G pseudotyped NL4-3.Luc.R-E- luciferase reporter viruses which were treated with DNAse (200 ng/ml) (Promega, Madison WI, USA) for 45 min at 37°C in medium containing 6 mM MgCl2 at a MOI of 0.1 and 1. Three hours post-infection the cells were washed with medium and the medium was replaced. pol proviral DNA copies and pol, Gag and GFP mRNA levels were analysed 48h post infection. Gag p24 levels in the culture supernatant were determined respectively 7 days post infection by an in-house enzyme-linked immunosorbent assay [28 (link)]. Luciferase activity was determined 48h post infection as a measure for HIV-1 replication, by adding 25 μl substrate (0.83 mM ATP, 0.83 mM D-luciferin (Duchefa, Haarlem, The Netherlands), 18.7 mM MgCl2, 0.78 μM Na2H2P2O7, 38.9 mM Tris pH 7.8, 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 μM dithiothreitol) directly to the culture medium. Luminescence was measured for 1 s per well using a luminometer (Berthold, Bad Wildbad, Germany).