Cells and viruses
MT2 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI1640, HyClone, Logan, UT) containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin (P/S, gibco, Waltham, MA). TZM-bl and HeLa cells were maintained in high-glucose Dulbecco’s Modified Eagle's medium (DMEM, HyClone, Logan, UT) containing 10% FBS and 1% P/S. All cell lines were incubated at 37 °C with 5% CO2. To produce the infectious HIV-1 AD8 strain, 20 μg pNL4.3(AD8) clone was transfected into HeLa cells using iN-fect™ (iNtRON Biotechnology, Seongnam, Korea). After 48 h of culture, culture media were harvested and centrifuged for 2000 rpm (1344 rcf) at 5 min for removal of cell debris. Harvested viruses were stored at − 80 °C. Infectious virus titers were determined based on 50% tissue culture infectious dose (TCID50) according to the endpoint method of Reed and Muench (1938).
RAL, DTG and EVG were kindly provided by the New Drug development team, R&D center, ST Pharm (Seoul, Korea) and BI 224436 by Professor Baek Kim (School of Medicine Health Science Research Building, Emory University, Atlanta, GA).
Measurement of cell cytotoxicity of anti-HIV drugs
To determine the cytotoxicity of anti-HIV drugs, cell viability was assessed via the water-soluble tetrazolium salt (WST) method using an EZ-Cytox kit (Daeil Lab Service, Seoul, Korea) according to the manufacturer’s instructions. Briefly, TZM-bl and MT2 cells were seeded on 96-well cell culture plates at a density of 1 × 104 cells/well and cultured overnight. Cells were treated with serial dilutions of each drug (two-fold dilutions from 50,000 to 2.54 nM). On days 2 and 5 of incubation, 10 μl EZ-Cytox solution was added to each well and incubated for 2 h, followed by spectrophotometric measurement of absorbance at 540 nm. The CC50 value of samples was defined as the concentration inducing 50% cell death.
Enzyme-linked immunosorbent assay for p24
ELISA was conducted to detect HIV-1 p24 for assessment of antiviral activity using a HIV-1 p24 ELISA kit (XpressBio, Frederick, MD). To this end, MT2 cells seeded on a 96-well cell culture plate at a density of a 1 × 104 cells/well were infected with 500 TCID50 of HIV-1 AD8 strain. Each drug was serially diluted threefold (from 10,000 to 0.51 nM) for treatment of cells. After five days of culture at 37 °C with 5% CO2, 100 μl of culture medium was harvested and supernatant obtained by centrifugation for 5 min at 5000 rpm (8400 rcf), with storage at − 80 °C. Cell culture media of non-infected and infected cells without INI treatment were set as the negative and positive control, respectively. The ELISA procedure was conducted according to the manufacturer’s instructions. Briefly, samples were mixed with 20 μl lysis buffer, 200 μl aliquots pipetted into a microplate, and incubated for 1 h at 37 °C. After incubation, the contents of the wells were aspirated and microtitration plates washed six times with 350 μl wash buffer. Each well was treated with 100 μl detection antibody for 1 h at 37 °C, which was subsequently removed by washing under the same conditions. An aliquot (100 μl) of streptavidin-HRP conjugate was added into each well, followed by incubation at room temperature for 30 min. After washing under the same conditions, 100 μl substrate solution was immediately dispensed into each well and incubated for 30 min at room temperature with protection from direct sunlight. For termination of the reaction, 100 μl stop solution was added to each well and absorbance values at 450 nm immediately read using a microplate reader.
Antiviral activity test using the TZM-bl cell system
TZM-bl cells were seeded on 96-well cell culture plates at a density of 1 × 104 cells/well and cultured overnight. Each drug was twofold serially diluted from 10,000 to 0.15 nM and treated to cells. After a 30 min incubation period, viral infection was performed with 500 TCID50 of HIV-1 AD8. Cells were cultured for 48 h after infection and luciferase activity measured using Beetle-Lysis Juice (PJK GmbH, Kleinblittersdorf, Germany) according to the manufacturer’s instructions. Briefly, the medium was removed, and cells washed three times with 200 μl PBS. Next, 100 μl Beetle-Lysis Juice containing luciferin and ATP were added to each well and incubated for 5 min with protection from sunlight. Subsequently, luciferase activity was measured using a micro beta counter (PerkinElmer, Waltham, MA) after transferring solutions to a white 96-well plate. Anti-HIV efficacy of drugs was determined based on reduced expression of luciferase relative to the virus-only treatment group.
Strand transfer inhibition assay
The strand transfer assay was performed using the HIV-1 integrase assay kit (XpressBio, Frederick, MD) according to the manufacturer’s instructions. Briefly, 100 μl of 1X donor substrate DNA (DS DNA) solution was added to each well and incubated for 30 min at 37 °C. The liquid was aspirated from the wells and washed 5 times with 300 μl wash buffer, followed by incubation with 200 μl blocking buffer per well for 30 min at 37 °C. Following aspiration of the liquid, wells were washed three times with 200 μl reaction buffer. Next, 100 μl IN enzyme solution was added to each well and incubated under similar conditions followed by removal of liquid and three washes with 200 μl reaction buffer. Each test sample was fivefold serially diluted (from 1000 to 8 μM) in reaction buffer and 50 μl aliquots added per well. After 5 min incubation at room temperature, 50 μl 1X target substrate DNA (TS DNA) solution was directly added to the 50 μl test sample within the wells. Reactions were mixed by tapping the plate gently 3–5 times and incubating for 30 min at 37 °C, washed 5 times with 300 μl wash solution and incubated with 100 μl HRP antibody for 30 min at 37 °C. After washing the plate under the same conditions, 100 μl TMB peroxidase substrate solution was added per well and incubated for 10 min at room temperature. To terminate the reaction, 100 μl TMB stop solution was directly added to wells and absorbance read using a plate reader at 450 nm.
Confirmation of inhibition of 3′-processing activity of NCINI
To confirm the 3′-processing inhibition activity NCINI, the strand transfer inhibition assay was modified. The plate coating process with DS DNA was conducted in a similar manner. However, prior to treatment with LTR DS DNA, aliquots of fivefold serially diluted INI (from 2000 to 3.2 μM) were incubated with 20 nM IN for 30 min. Reaction of integrase first with the drug before its reaction with DS DNA is a necessary step to validate the 3′-processing inhibitory activity of the compound. The integrase-inhibitor mixture was added to LTR DS DNA-conjugated 96-well plates. Subsequent steps were conducted in a similar manner as the strand transfer assay.
All measures of variance are presented as standard error of mean (SEM). Data were analyzed via two-way analysis of variance (two-way ANOVA) with Tukey post-hoc test using Prism8 (GraphPad Software, San Diego, CA). Differences were considered significant at p-values < 0.05.