Dissecting the mode of action of various HIV-inhibitor classes in a stable cellular system

Abstract

We describe a stable and sensitive HIV evaluation system, which discriminates HIV-specific membrane fusion and early transcription events and is suitable for high-throughput inhibitor screening. A human lymphocytic line, constitutively producing infectious HIV-1, serves as Env-positive donor. A second indicator cell line carries a silent HIV-1 LTR lacZ reporter plasmid. A bicellular cocultivation setup allows titration and standardization of "fusion-induced gene stimulation (FIGS)" events. With few manipulations aspects of fusion and/or LTR induction can be distinguished and simultaneously assayed. Anti-Env-V3 antibodies prevent fusion and subsequent lacZ induction, and a Tat-specific inhibitor blocks only lacZ induction in a dose dependent manner without affecting membrane fusion. The LTR reporter is readily activated by Tat from HIV-1, HIV-2, or SIV and it responds to exogenous Tat protein. The reporter system is sensitive enough to detect single infection events on pre-seeded layers of indicator cells, which renders it potentially useful for direct virus quantification in patients' material. Moreover, our system allows to control and normalize DNA transfection efficiencies of HIV-derived plasmids. This aspect is particularly valuable for studies of RT- and protease-inhibitors and resistances, where p24 or supernatant reverse transcriptase, otherwise standard virus readouts, can be directly affected by inhibitors or mutations.

TLR7/8 Triggering Exerts Opposing Effects in Acute versus Latent HIV Infection

“…We used a cell-based fusion assay with HeLa cells expressing gp140 from LAI and the HIV transactivator tat (HeLa-Env/LAI) (17) and HeLa cells expressing CD4 and the LTR-driven lacZ gene (HeLa SX CCR5) (18). Fusion of HeLa-Env/LAI cells and HeLa SX CCR5 results in transcription of the lacZ gene.…”

A Neutravidin-based Assay for Reverse Transcriptase Suitable for High Throughput Screening of Retroviral Activity

“…Radioactive RT assay The radioactive RT assay was performed as previously described (Klimkait et al, 1998). In brief, the assay utilised 32 P-labelled dTTP (NEN) and oligo d(T) in a reaction mix that, after 2 h incubation at 37 o C was dot blotted onto DEAE paper.…”

“…Approximately 0.2 µg cloned DNA was transfected into SX-22-1 HeLa cells (Klimkait et al, 1998) using Lipofectamine 2000 according to the protocol supplied by the manufacturer (Life Technologies, Basel, CH). Cells were subsequently incubated in fresh complete DMEM-medium, containing HIV inhibitor at the indicated concentration, for 48 h prior to collection of cells and viral supernatants.…”

“…Cells were subsequently incubated in fresh complete DMEM-medium, containing HIV inhibitor at the indicated concentration, for 48 h prior to collection of cells and viral supernatants. For immunoblot analysis, cells were scraped from the plates and lysed as previously described (Klimkait et al, 1998).…”

“…infections Transfection supernatants were normalised for the number of viral particles present using transfection efficiency as a quantifiable marker (Klimkait et al, 1998). For infection cultures, transfection supernatant was added to 1mL 8 × 10 4 CEM-SS cells in a 24-well plate.…”