Gal screen β galactosidase reporter gene assay system for mammalian cells

ELVIS^TM cells are genetically engineered baby hamster kidney cells that encode a lacZ gene, which is expressed upon infection via the viral transactivator ICP10 (Enzyme-Linked Virus-Inducible System – ELVIS^TM) (Proffitt and Schindler, 1995 (link)). 5,000 ELVIS cells were seeded into 96-well plates and mixed with 10 μl of the reconstituted fractions of the placenta library. After 10 min, cells were infected with a clinical HSV-2 isolate (Krüger et al., 2019 (link)) and infection was determined 2 days later by detecting the β-galactosidase activity in cellular lysates using the Gal-Screen β-Galactosidase Reporter Gene Assay System for Mammalian Cells (Thermo Fisher Scientific) and the Orion II microplate luminometer (Berthold, Bad Wildbad, Germany). All values represent reporter gene activities derived from triplicates minus background activities derived from uninfected cells. Triplicates are expressed as mean ± standard error derived from two independent experiments. All experiments with infectious viruses were performed in a BSL3^∗∗ laboratory in accordance with biosafety guidelines by Ulm University Hospital.

Virus stocks of CCR5-tropic HIV-1 NL4-3 were generated by transient transfection of HEK293T cells with proviral DNA as described (Münch et al., 2007 (link)). Transfection mixture was replaced by 2 ml DMEM supplemented with 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin and 2.5% heat-inactivated FCS after 8 h of incubation. 48 h later virus was collected by centrifuging the cell supernatant to remove cell debris for 3 min at 300 g. Virus stocks were stored at –80°C. For the infection assay, 10.000 TZM-bl cells were seeded the day before into 96-well-flat-bottom plates. Before infection, 80 μl of cell medium was added to the wells. Virus treatment experiments were done by mixing 70 μl of the peptide sample with 70 μl of 1/20 diluted HIV-1 for 1 h at 37°C. Then, 40 μl of the peptide-virus mix were added to each well. Two days post infection, the rates of infection were measured by Gal−Screen β−Galactosidase Reporter Gene Assay System for Mammalian Cells (Thermo Fisher Scientific) and the Orion II microplate luminometer (Berthold Technologies GmbH & Co., KG, Bad Wildbad, Germany). Values were corrected for the background signal derived from uninfected cells and antiviral effect of the peptide was then calculated by normalization to untreated cells which were set as 100% infection.

HIV-1 infection was quantified using TZM-bl reporter cells stably transfected with an LTR-lacZ cassette (Wei et al., 2002 (link)). Upon infection with HIV-1 the viral protein Tat is expressed, which activates the LTR promotor and results in β-galactosidase expression. For infection assays, 10,000 TZM-bl cells were seeded the day before into 96-well plates. The next day, the medium was replaced with serumfree X-vivo 15 medium (Lonza, BE02-060F). For cell treatment, cells were treated with the titrated compounds and afterward infected. For virus treatment, virus and titrated compounds were mixed and preincubated and subsequently added onto the cells. 2 days later, infection rates were determined by detecting the β-galactosidase activity in cellular lysates using the gal-screen β-galactosidase reporter gene assay system for mammalian cells (Thermo Fisher Scientific) and the Orion II microplate luminometer (Titertek Berthold). Measured values represent reporter gene activity (RLU/s) and were corrected for the background signal derived from uninfected cells. Untreated controls were set to 100% infection.