PERMEABILIZATION OF MAMMALIAN-CELLS TO PROTEINS - POLIOVIRUS 2A(PRO) AS A PROBE TO ANALYZE ENTRY OF PROTEINS INTO CELLS

Two hybrid protein molecules containing the poliovirus protease 2A (MB P-2A(pro)) (maltose-binding protein-2A(pro) and MBP-Pseudomonas exotox in A-2A(pro)) have been constructed and purified. Both hybrid proteins efficiently cleave the translation initiation factor eIF-4G when they are co-internalized into cells with adenovirus particles. Almost no i ntact eIF-4G can be detected in cells incubated with these proteins fo llowing this method. Reovirus infectious subviral particles also promo te the delivery of MBP-2A(pro) into cells, although less efficiently t han adenovirus particles. None of the other methods employed to permea bilize cells to MBP-2A(pro) achieves the degree of eIF-4G cleavage obs erved with adenovirus particles. By comparison about 30% of cells elec troporated with MBP-2A(pro) still contain intact eIF-4G. More drastic electroporation conditions lead to a significant decrease of cell surv ival. Osmotic lysis of pinocytic vesicles resulted in 30% of the eIF-4 G being cleaved in cells treated in suspension. Delivery of MBP-2A(pro ) by pH-sensitive Liposomes leads to poor hydrolysis of eIF-4G. Taken together our results indicate that permeabilization of cells with aden ovirus particles is the most efficient method for introducing MBP-2A(p ro) into cells. (C) 1997 Academic Press.