In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication

Lainé, Sébastien; Scarborough, Robert J.; Lévesque, Dominique; Didierlaurent, Ludovic; Soye, Kaitlin J; Mougel, Marylène; Perreault, Jean‐Pierre; Gatignol, Anne

doi:10.4161/rna.8.2.15200

Cited by 16 publications

(13 citation statements)

References 44 publications

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“…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. The bands were visualized as described 86 and quantified by densitometry analysis, using Adobe Photoshop CS3 10.0.1 software.…”

Section: Rt-pcrmentioning

confidence: 99%

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

et al. 2015

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Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and RevResponse Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

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Section: Rt-pcrmentioning

confidence: 99%

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

et al. 2015

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“…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).…”

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confidence: 99%

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

Scarborough

Adams

Daher³

et al. 2015

Antimicrob Agents Chemother

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We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection.

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“…The Gag coding sequence had much lower overall conservation; however, four highly conserved and, with some exceptions to our conservation criteria ( Figure 2a ), nine moderately conserved target sites were identified in this region ( Figure 2c ). Of the Rzs that we have previously evaluated targeting the Tat/Rev exon1 coding sequence of HIV-1 RNA, 16 the target sites for Tat1 and Tev1 were highly and moderately conserved, respectively. Conservation exceptions and sequences of all target sites used in this study are illustrated, along with the DNA sequences of the corresponding SOFA-HDV-Rz variable regions, in Supplementary Table S1 .…”

Section: Resultsmentioning

confidence: 99%

“…21,22 HIV-1 RT activity was measured to estimate the production of virus released into the medium of transfected cells and effects of Rzs were normalized to cotransfection of pNL4-3 with an empty Rz expression plasmid. An irrelevant Rz targeting Hepatitis B Virus RNA (SOFA-HDV-RzHBV, adapted from SOFA-δRz-303 9 ) was used as a negative control and previously described SOFA-HDV-Rzs Tat1 and Tev1 16 were used as positive controls. Compared to SOFA-HDV-RzHBV, Rzs targeting both the 5′UTR and Gag coding sequences significantly inhibited viral production, with the top candidate (SOFA-HDV-Rz-1498) targeting the Gag coding sequence ( Figure 3 ).…”

Section: Resultsmentioning

confidence: 99%

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

Scarborough

Lévesque

Boudrias-Dalle

et al. 2014

Molecular Therapy - Nucleic Acids

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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.

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In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication

Cited by 16 publications

References 44 publications

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

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

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

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

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