Fig. 2

Determination of the mode of action of BPPT. A Time-of-addition assay. TZM-bl cells (1 × 10⁴) were infected with HIV-1_NL4-3 at an MOI of 0.5. Subsequently, the cells were treated with the compounds at the indicated time points. At 48 h postinfection, the viral activity was measured as the F-Luc activity using the Bright-Glo luciferase assay kit. The relative activities are expressed as the mean ± SD (n = 3) compared with that in the DMSO control measured after 48 h of infection. The time frames associated with each HIV-1 infection step are represented based on susceptibility to the well-known drugs. RT: reverse transcription, IN: integration. B and C. HEK293T cells (1.5 × 10⁵) were transfected with 200 ng of the pBR43IeG-nef⁺ plasmid. Subsequently, the cells were treated as described for Fig. 1C. After 48 h, the GFP-expressing cells were observed using fluorescence microscopy (× 200 magnification) (B), the count of GFP-positive cells was determined using flow cytometry (B), and the amount of viral production was measured using the p24 ALPHALISA™ kit (C). ***p < 0.001, **p < 0.01, and *p < 0.05, compared with the cells treated with DMSO. n.s.: non-significant. D An in vitro HIV-1 RT assay was performed with BPPT, EFV, and NVP, as described in “Materials and Methods.” E An in vitro HIV-1 integrase assay was performed with BPPT (10 μM), EFV (10 μM), and raltegravir (RAL, 10 μM), as described in “Materials and Methods.” Data are expressed as the mean ± SD (n = 3) compared with the DMSO control. F A3.01 cells (1 × 10⁶) infected with DNase I-treated active or heat-inactivated HIV-1_NL4-3 at an MOI of 1 were treated with the indicated compounds (1 μM). After 6 h of infection, viral reverse transcription (RT) product (viral cDNA) was extracted; its level was determined using quantitative real-time PCR (RT-qPCR) and represented as the mean ± SD (n = 3) normalized against β-globin compared with that in cells treated with DMSO. G A3.01 cells (1 × 10⁶) infected with DNase I-treated HIV-1_NL4-3 at an MOI of 1 were treated with the indicated compounds (1 μM). After 24 h of infection, the level of integrated viral DNA was determined using Alu-PCR, as described in “Materials and Methods.” The relative activity is expressed as the mean ± SD (n = 3) compared with the DMSO control. C and E–G ***p < 0.001, compared with the DMSO control. n.s.: non-significant