DISSECTING THE MODE OF ACTION OF VARIOUS HIV-INHIBITOR CLASSES IN A STABLE CELLULAR-SYSTEM

We describe a stable and sensitive HN evaluation system, which discrim inates HIV-specific membrane fusion and early transcription events and is suitable for high-throughput inhibitor screening. A human lymphocy tic line, constitutively producing infectious HIV-1, serves as Env-pos itive donor. A second indicator cell line carries a silent HIV-1 LTR l acZ reporter plasmid. A bicellular cocultivation setup allows titratio n and standardization of ''fusion-induced gene stimulation (FIGS)'' ev ents. With few manipulations aspects of fusion and/or LTR induction ca n be distinguished and simultaneously assayed. Anti-Env-VS antibodies prevent fusion and subsequent lacZ induction, and a Tat-specific inhib itor blocks only lacZ induction in a dose dependent manner without aff ecting membrane fusion. The LTR reporter is readily activated by Tat f rom 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. Moreove r, our system allows to control and normalize DNA transfection efficie ncies of HIV-derived plasmids. This aspect is particularly valuable fo r 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.