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 cellular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphtheria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation signal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in cells expressing the viral protease in the cytosol, i.e. virus-infected cells, thereby providing a specific eradication of these cells. In the present work 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 PR exclusively at the introduced sites, and thereby are converted from unstable to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, the toxins were unable to selectively eradicate HIV-1-infected cells, apparently due to low cytosolic HIV-1 PR activity, since we could not detect cleavage of the toxins by HIV-1 PR in infected cells. Alternative strategies for the construction of toxins that can specifically be activated by viral proteases are discussed.
Falnes
P O
PO
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. pfalnes@ulrik.uio.no
Welker
R
R
Kräusslich
H G
HG
Olsnes
S
S
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000235:genetics / Q000493:pharmacokinetics* / Q000494:pharmacology; D001483:Base Sequence; D001711:Biotransformation; D017931:DNA Primers; D004167:Diphtheria Toxin / Q000235:genetics / Q000493:pharmacokinetics* / Q000494:pharmacology; D016333:HIV Protease / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D017931:DNA Primers; D004167:Diphtheria Toxin; D011500:Protein Synthesis Inhibitors; C030325:anthrax toxin; D016333:HIV Protease