Inhibition Of Rev Research Articles

Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen

New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE–Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs.

Functional role of the HIV-1 Rev exon 1 encoded region in complex formation and trans-dominant inhibition

To study functional aspects of the exon 1 encoded region of the human immunodeficiency virus type 1 Rev protein, the viral Tev protein which exhibits low Rev activity but lacks the rev exon 1 encoded region was examined. Neither Rev–Tev heteromer complex formation nor inhibition of Rev by an export deficient Tev mutant was observed. Insertion of the rev exon 1 encoded region into the Tev mutant allowed it to oligomerize with Rev and act as a trans-dominant negative mutant. This showed that the exon 1 encoded region of Rev is essential for oligomerization and that oligomerization is a prerequisite for trans-dominant inhibition.

Inhibition of rev·RRE complexation by triplex tethered oligonucleotide probes

We have described a class of molecules, called tethered oligonucleotide probes (TOPs), that bind RNA on the basis of both sequence and structure. TOPs consist of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. In a triplex TOP, one oligonucleotide recognizes a short single-stranded region in a target RNA through the formation of Watson-Crick base pairs; the other oligonucleotide recognizes a short double-stranded region through the formation of Hoogsteen base pairs. Binding of triplex TOPs to an HIV-1 Rev Response Element RNA variant (RRE AU) was measured by competition electrophoretic mobility shift analysis. Triplex TOP·RRE AU stabilities ranged between −9.6 and −6.1 kcal mol 1 under physiological conditions of pH, salt, and temperature. Although the most stable triplex TOP·RRE AU complex contained 12 contiguous U·AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Triplex TOPs inhibited formation of the RRE·Rev complex with IC 50 values that paralleled the dissociation constants of the analogous triplex TOP·RRE AU complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of Rev·RRE AU complexation by triplex TOPs did not require pre-incubation of RRE AU and a TOP: triplex TOPs competed efficiently with Rev for RRE AU and inhibited RRE AU·Rev complexation at equilibrium.

Inhibition of the HIV Rev transactivator : a new target for therapeutic intervention.

The viral transactivator Rev is essential for HIV replication, since it allows the nuclear export of unspliced and partially spliced viral mRNAs that encode the structural proteins. Rev is an RNA binding protein that interacts with a highly structured RNA element, the RRE, found within the envelope sequences. This viral protein also interacts with cellular proteins, termed nucleoporins, and acts as an adaptor between the viral mRNAs and the cellular nuclear export machinery. Both interactions are specific, and required for Rev function. Because of its crucial role in the HIV replication cycle, and its novel mechanism of action, Rev represents an ideal target for therapeutic intervention. This review describes the efforts towards Rev inhibition. Gene therapy approaches, including the expression of trans-dominant mutants and RNA decoys, as well as antisense therapies and small molecule inhibitors of Rev-RRE binding or Rev interaction with the cellular machinery will be discussed