The assay described in this paper is based on the expression of
the PR/Gal4 fusion in an inducible manner via a Tet-On system
(adapted from Clontech), thus drastically reducing the possible
toxic side-effects of PR. The reverse tetracycline transactivator
(rtTA) used in this system allows for the induction of PR/Gal4
expression by the addition of tetracycline (Tet) or doxycycline
(Dox), only when needed. eGFP, the sensor for PR activity, will be
expressed only when PR/Gal4 expression is induced in the
presence of a PI. Most importantly, all the assay elements have
been stably expressed in mammalian cells using retroviral
technology. Finally, we established T-cell lines from clones with
the highest sensitivity showing robust, reliable and reproducible
behavior. This is the first time a cell-based assay for monitoring
PR activity has been developed in T-cells, the natural milieu of
HIV infection, thus providing an efficient way to search for novel
inhibitors/competitors of PR, which could lead to the development
of new therapeutics against HIV.