Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells.

Abstract

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.

Oligodeoxynucleotide analogs with 5′‐linked anthraquinone

“…The selection of methylphosphonate (P-CH3) [3,4] or phosphorothioate (P-S) [5,6] modified ODNs to provide nuclease resistance has greatly enhanced the possibility that such antisense ODNs could be used in vivo. Indeed, an antisense effect has been shown to occur in vitro for HIV infection in chronically infected T cells [7]. Similar antisense effects have been reported with normal and S-ODNs in cancer ceils in which oncogenes are overexpressed [8,9], although complete inhibition of expression was not achieved.…”

“…Measurements of Tm values indicate that intercalation of the anthraquinone occurs with both linkers (table 1). Specific base sequences that were synthesized with 5'-linked anthroquinone are: dT1, dT4, dT8, dT12, dTl~, dC15, and anti-sense phosphorothioate oligos" c-myc 15-mer [8,9] and the anti-rev (S-cr-rev) HIV sequence: d(5 '-AQ-TCG TCG CTG TCT CCG CTT CTT CCT GCC A) [7]. The anthraquinone-ODNs described here exhibited anti-HIV activity in an in vitro assay system [31], and can be expected to show other antiviral properties.…”

“…Similar antisense effects have been reported with normal and S-ODNs in cancer ceils in which oncogenes are overexpressed [8,9], although complete inhibition of expression was not achieved. In these cases, the S-ODNs were not found to be toxic to the cells, although some of the normal ODNs were somewhat toxic, presumably due to nuclease digestion to mononucleotides [7,10].…”

Antisense oligonucleotides in solution or encapsulated in immunoliposomes inhibit replication of HIV-1 by several different mechanisms

“…These include phosphorothioate oligonucleotides and oligonucleotides with the sugar moiety oriented in the at configuration. Phosphorothioate oligonucleotides have been shown to inhibit the replication of HIV-1 in vitro by both sequence-non-specific (3,5,10,11) and specific processes (12)(13)(14)(15)(16), depending on the type of infection. In acutely infected cells, phosphorothioate oligonucleotides acted in a sequence-non-specific fashion probably by inhibition of viral binding and fusion (17,18) and/or viral reverse transcriptase (RT) (19,20).…”

Transdominant Rev and Protease Mutant Proteins of HIV/SIV as Potential Antiviral Agents In vitro and In vivo.

“…Since both tat and rev are essential regulatory genes, they are, along with the viral enzymes, suitable targets for anti-viral strategies. The rev gene is a particularly attractive target because of our previous finding that an incomplete inhibition of rev could nonetheless result in complete block of Gag/Pol protein expression (Matsukura, et al, 1989), suggesting that virus expression is extremely sensitive to rev depletion.…”