Toxins that are activated by HIV type-1 protease through removal of a signal for degradation by the N-end-rule pathway

Diphtheria toxin enters the cytosol of mammalian cells where it inhibits ce llular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphth eria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation sig nal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in ce lls expressing the viral protease in the cytosol, i.e. virus-infected cells , thereby providing a specific eradication of these cells. In the present w ork we describe the construction of toxins that contain a signal for N-end- rule-mediated degradation just upstream of a cleavage site for the protease from HIV type 1 (HIV-1 PR). We show that the toxins are cleaved by HIV-1 P R exclusively at the introduced sites, and thereby are converted from unsta ble to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, t he toxins were unable to selectively eradicate HIV-1-infected cells, appare ntly due to low cytosolic HIV-1 PR activity, since we could not detect clea vage of the toxins by HIV-1 PR in infected cells. Alternative strategies fo r the construction of toxins that can specifically be activated by viral pr oteases are discussed.