SPECIFIC-INHIBITION OF INFLUENZA-VIRUS RNA-POLYMERASE AND NUCLEOPROTEIN GENES EXPRESSION BY LIPOSOMALLY ENDOCAPSULATED ANTISENSE PHOSPHOROTHIOATE OLIGONUCLEOTIDES - PENETRATION AND LOCALIZATION OF OLIGONUCLEOTIDES IN CLONE-76 CELLS

Liposomally encapsulated phosphorothioate oligonucleotides with four t arget sites (PB1, PB2, PA, and NP) were synthesized and tested for inh ibitory effects by a CAT-ELISA assay using the clone 76 cell line. The liposomally encapsulated phosphorothioate oligonucleotides (S-ODNs) c omplementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. Displacement of the target AUG initi ation codon sequence to the 3'-end, 5'-end, and/or center sites on the antisense phosphorothioate oligonucleotides was studied with regard t o the inhibition of influenza virus RNA polymerases and NP. The antise nse phosphorothioate oligonucleotide containing the AUG initiation cod on at the center site of the oligonucleotide had the highest inhibitor y effects. The liposomally encapsulated phosphorothioate oligonucleoti des exhibited higher inhibitory activity than the free oligonucleotide s. Observation of clone 76 cells treated with the endocapsulated antis ense phosphodiester oligonucleotide, FITC-ODNs-PB2-T3, by a confocal l aser scanning microscope, revealed diffuse fluorescence, apparently wi thin the cytoplasm. Interestingly, the endocapsulated antisense phosph orothioate oligonucleotide, FITC-S-ODNs-PB2-T3 accumulated in the nucl ear region of clone 76 cells. However, weak fluorescence was observed in the endosomes and in the cytoplasms of the clone 76 cells treated w ith the free antisense phosphorothioate oligonucleotides. (C) 1997 Aca demic Press.