In this report, we demonstrate the sequencespecific suppression of viral expression in T cells chronically infected with human immunodeficiency virus 1 (HIV-1), using antisense phosphorothioate oligodeoxynucleotides. As a target for antisense intervention, we used the HIV-1 gene rev, which is essential for viral replication and regulates the expression of virion proteins, in part, by affecting the splicing of the viral mRNA. A phosphorothioate oligomer complementary to the initiation sequence of HIV-1 rev had a significant and selective inhibitory effect on the production of several viral proteins in chronically HIV-1-infected T cells and drastically reduced the unspliced (genomic) viral mRNA transcripts, with relative sparing of smaller (spliced)
METHODS AND MATERIALSSynthesis and Purification of Phosphorothioate and Unmodified Normal Oligo(dN). The phosphorothioate oligomers were synthesized by the method previously reported (6). Unmodified normal oligomers were synthesized by the standard method. All syntheses were performed with an automated DNA synthesizer (Applied Biosystems; model 380-B).Sequences of the Target Region in the HIV Genome and Oligomers Used. We used the rev gene (formerly called art/trs) as a target for antisense intervention of viral gene expression. The rev gene is well conserved among HIV-1 clones. To clarify the sequence specificity, we tested an unmodified antisense oligomer of rev (normal phosphodiester linkages; n-arev) and phosphorothioate oligomers containing rev sense sequence (S-sense-rev), rev antisense sequence (S-arev), random sequence with the same base composition as S-arev (S-random-arev), 28-mer homooligomer oligo(dC)28 (S-dC28), rev antisense sequence containing four N3-methylthymidine (N-MedThd) residues (S-N-Me-arev), and an antisense sequence against the initiation site of gag, the gene encoding the group-specific antigen gag (S-agag) (Fig. 1). Viral Gene Expression Inhibition Assay. To test whether an agent has regulatory activity on HIV viral expression, we used chronically HIV-1-infected H9 cells, which were exposed to the virus isolate HTLV-IIIB, kept in culture for several months or more in complete medium (RPMI 1640 supplemented with 15% fetal calf serum, 4 mM L-glutamine, 50 nM 2-mercaptoethanol, and 50 units of penicillin, and 50 ,ug of streptomycin per ml N3-methylthymidine; n-arev, nor-mal (unmodified) antisense oligo(dN) against rev; S-dC28, 28-mer phosphorothioate oligo(dC); S-agag, antisense phosphorothioate oligo(dN) against gag; S-arev, antisense phosphorothioate oligo(dN) against rev; S-N-Me-arev, antisense phosphorothioate oligo(dN) against rev containing four N-MedThd residues in the sequence; S-random-arev, phosphorothioate random oligo(dN) with the same base composition as S-arev; S-sense-rev, phosphorothioate sense oligo(dN) of rev; RIPA, radioimmunoprecipitation assay.