A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

Abstract

Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

“…34,83,84 For virus production, HEK 293T cells were transfected with HIV-1 provirus pNL4-3 and supernatants were harvested 48 h post-transfection. Viral content of supernatant was evaluated by performing a reverse-transcriptase assay as described.…”

“…Viral content of supernatant was evaluated by performing a reverse-transcriptase assay as described. 84 For the infection, volumes of supernatant corresponding to 1 or 2 million cpms of reverse transcriptase activity were applied to Jurkat cells for 3 h before washing.…”

“…Cell lysates were prepared, separated and transferred for immunoblotting as previously described. 34,83,84 Membranes were blocked for 1 h in 5% nonfat milk and 0.05% Tris-buffered saline (TBS)-Tween and incubated overnight at 4 C with anti-EGFP (Santa Cruz) antibody at a 1/1000 dilution, anti-Myc (Santa Cruz) antibody at a 1/1000 dilution, anti-actin (Chemicon) antibody at a 1/ 10,000 dilution, anti-Dicer349 5 or anti-Dicer (Medimabs, Montreal, QC, CA) at a 1/5000 dilution, anti-TRBPjbx 36 at a 1/500 dilution, anti-Ago2 7C6 (from Dr. T. Hobman) at a 1/5000 dilution, anti-HIV-1 p24 183-H12-5C 83,84 at a 1/1000 dilution, anti-Ad2/5 E1A (Santa Cruz) at a 1/1000 dilution, or anti-GAPDH (Santa Cruz) at a 1/1000 dilution in 5% milk/TBST. After 5 washes in TBST, membranes were incubated with peroxidase-conjugated secondary donkey anti-rabbit antibody (Amersham) for TRBP, Ago2, Dicer and E1A, and sheep antimouse (Amersham) for EGFP, Myc, Actin, p24 and GAPDH at a 1/5000 dilution.…”

Effective Inhibition of HIV-1 Production by Short Hairpin RNAs and Small Interfering RNAs Targeting a Highly Conserved Site in HIV-1 Gag RNA Is Optimized by Evaluating Alternative Length Formats

“…We previously screened HIV-1 RNA for conserved ribozyme target sites (35,36) based on the specific on/off adaptor (SOFA) hepatitis delta virus (HDV) ribozyme motif (37)(38)(39). A target site in the Gag coding sequence was identified that was highly conserved and accessible to a ribozyme and an shRNA (36).…”

Novel Prospective Treatment Options

“…The third solution involves identifying novel, genetically conserved sequences of HIV which do not usually undergo mutation. Targeting these stable sites would favour the success of RNAi [114]. Finally, RNAi techniques are also being designed to restrict the 'genetically more stable' host factors that help in HIV replication (discussed later under strategies to enhance target cell resilience to HIV infection).…”