ANTIVIRAL ACTIVITY AND PROTECTION OF CELLS AGAINST HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 USING AN ANTISENSE OLIGODEOXYRIBONUCLEOTIDE PHOSPHOROTHIOATE COMPLEMENTARY TO THE 5'-LTR REGION OF THE VIRAL GENOME

A COS-like monkey kidney cell line stably transfected with the plasmid s pCMV gagpol-rre-r with the gag and pol genes, and pCMV rev with the rev gene of HIV-1 derived from the cDNA clone BH10, was used as a mode l for assessing the effectiveness of antisense (AS) constructs. A 20-m er oligodeoxyribonucleotide (oligo) phosphorothioate sequence (5'-CCG CCC CTC GCC TCT TGC CG) complementary to a portion of the 5'-long term inal repeat (5'-LTR) of the HIV-1 genome was tested for its inhibitory effects on the biologically important processes of HIV-1 replication and proliferation. We observed a concentration-dependent inhibition of HIV protein synthesis. Desitometric analysis of data from Western blo t analysis showed sequence-specific and concentration-dependent oligo inhibition of p24 viral core antigen formation in the low-mu M range. When lipofectin was used as a delivery vehicle, a markedly increased p otentiation of the AS activity of the sequence was observed at a lower concentration (0.1 mu M), following a 24-h preincubation. The AS cons truct specifically inhibited intracellular p24 production in chronical ly HIV-1-infected cells of lymphoid origin (H9/IIIB cells) by 95%, res ulting in a 15-fold inhibitory effect relative to a similar sequence t hiolated at only seven single-base positions, A concentration-dependen t attenuation in the reverse transcriptase activity and a reduction in viral p24 level was observed in the culture supernatant of AS-pretrea ted HIV-1-infected phytohemagglutinin A-stimulated human cord blood mo nonuclear cells. Incubation of a HIV-1-infected lymphoid cell line wit h AS sequence resulted in a marked reduction in syncytium formation, a nd therefore protected cells from the cytopathic effects of the virus. Furthermore, the AS oligo did not appear to be cytotoxic in cell grow th rate and colony-forming ability assays. The AS oligo described in t his report is a useful new tool for the molecular analysis of HIV-1 ge ne expression and proliferation, and may have potential as a therapeut ic agent.