INDUCTION OF TOXIN SENSITIVITY IN INSECT CELLS BY INFECTION WITH BACULOVIRUS ENCODING DIPHTHERIA-TOXIN RECEPTOR

The diphtheria toxin receptor (DTR) has been identified as the precurs or of heparin-binding epidermal growth factor-like growth factor, whic h may interact with other membrane proteins to form the functional rec eptor. To test if mammalian DTR is able to confer toxin sensitivity on to phylogenetically distant cells, we expressed monkey DTR in the bacu lovirus system and tested infected insect cells for toxin sensitivity. cDNA encoding an epitope-tagged heparin-binding epidermal growth fact or-like growth factor precursor (DTR(B3)) was inserted into the virus genome by allelic replacement to construct the recombinant virus VAc-D TR(B3). SF9 cells infected with vAc-DTR(B3), expressed functional DTR, which could be precipitated from the solubilized membrane fraction of infected cells with Sepharose-immobilized diphtheria toxin. The highe st level of expression (about 5 x 10(6) receptors/cell) was observed 4 8 h after infection, at which time the infected cells were highly sens itive to diphtheria toxin. Uninfected SF9 cells and cells infected wit h the wild type virus were resistant to the toxin. The presence of hep arin increased both the binding and the toxin sensitivity of vAc-DTR(B 3)-infected SF9 cells. Translocation of toxin A fragment was induced w hen cells with surface bound toxin were exposed to low pH, and the tra nslocation was optimal at pH less than or equal to 5.5. It was similar to 100 times more efficient at 24 degrees C than at 4 degrees C. The data indicate that monkey DTR is fully functional when expressed in in sect cells.