Steady glo luciferase

To assess viral infectivity, 8 × 10⁵ SERINC3 KO 293T cells were seeded on 6 well plates and transfected with 2 μg pNL4.3ΔNef, 30 ng pSELECT-SERINC3(iHA)-ΔGFP or pSELECT-SERINC5(iHA)-ΔGFP and 10 ng pSELECT-nef-GFP using DNAfectin 2100 (Applied Biological Materials). Culture supernatants containing viruses were harvested at 48-hours post-transfection, centrifuged for 10 minutes at 1000 x g to pellet residual cells or debris and then stored as aliquots at -80°C until use. Viral quantification was done using p24 ELISA (XpressBio). A standardized amount of each virus (5 pg or 10 pg p24) was used to infect 1 x 10⁴ TZM-bl reporter cells in a 96-well flat bottom plate in triplicate. Infectivity was measured at 48 hours using Steady-Glo Luciferase (Promega) on a Tecan Infinite M200 PRO plate reader.

A SARS-CoV-2 pseudovirus expressing a luciferase reporter gene was generated in an approach similar to that described previously (10 (link), 28 (link), 29 (link)). Briefly, the packaging construct psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and S protein expressing pcDNA3.1-SARS CoV-2 S.dCT were cotransfected into HEK293T cells using lipofectamine 2000 (Thermo Fisher Scientific). After 48 h, supernatant was collected, and pseudotyped viruses were purified by filtration with a 0.45-μm filter. To determine the neutralization activity of the antisera from vaccinated animals, HEK293T-hACE2 target cells were seeded in 96-well tissue culture plates at a density of 1.75 × 10⁴ cells per well and incubated overnight. Then, 3-fold serial dilutions of heat-inactivated serum were prepared and mixed with 50 μl of pseudovirus. The mixture was incubated at 37°C for 1 h before being added to HEK293T-hACE2 cells. Then, 48 h after infection, cells were lysed in Steady-Glo luciferase (Promega) according to the manufacturer’s instructions. Neutralization titers were defined as the sample dilution at which a 50% reduction in relative light units was observed relative to the average of the virus control wells.

A Jurkat latency model derived from Jurkat clone E6-1 cells (ATCC, TIB-152), as described in detail elsewhere (4 (link)), containing a 1:1:1 ratio of 3 different clones (C16, I15, and N6), which were selected for HIV expression quiescence but inducibility, was used to conduct a targeted screen for combination latency reversal regimens. Each clone has a luciferase reporter in place of nef, and the N6 clone has an additional cell-surface murine HSA reporter. Cells were maintained as described previously, and dose-response curves of various LRAs dissolved in DMSO were cross titrated in a 10 × 10 grid against a dose-response curve of IAPi using a D300e Digital Dispenser (Hewlett-Packard). All conditions were normalized to 0.5% (v/v) DMSO. Following incubation for the time periods indicated, the viral luciferase reporter luminescence was measured using Steady-Glo Luciferase (Promega) and an EnVision 2102 Multilabel Plate Reader (PerkinElmer) according to the manufacturer’s instructions. Luminescence values were normalized to percentage of the background luminescent signal from DMSO-treated cells run on each plate.