Inhibition of hepatitis C virus replication and internal ribosome entry site-dependent translation by an RNA molecule

Abstract

Hepatitis C virus (HCV) protein synthesis is mediated by a highly conserved internal ribosome entry site (IRES), mostly located at the 59 untranslatable region (UTR) of the viral genome. The translation mechanism is different from that used by cellular cap-mRNAs, making IRESs an attractive target site for new antiviral drugs. The present work characterizes a chimeric RNA molecule (HH363-50) composed of two inhibitors: a hammerhead ribozyme targeting position 363 of the HCV genome and an aptamer directed towards the essential stem-loop structure in domain IV of the IRES region (which contains the translation start codon). The inhibitor RNA interferes with the formation of a translationally active complex, stalling its progression at the level of 80S particle formation. This action is likely related to the effective and specific blocking of HCV IRES-dependent translation achieved in Huh-7 cells. The inhibitor HH363-50 also reduces HCV RNA levels in a subgenomic replicon system. The present findings suggest that HH363-50 could be an effective anti-HCV compound and highlight the possibilities of antiviral agents based on RNA molecules.

Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

“…Fourteen cycles of selection-amplification were performed, and the selective pressure was stepwise modified by increasing the binding temperature from round IV on, and by reducing the target:aptamer ratio from round XI on. The yield of complex formation increased along the selection process, as shown by the evolution of B max 32 (Fig. 2).…”

“…The evaluation of the binding efficiency of the selected aptamers was essentially performed as previously described32, using trace amounts of renatured, 5′ end 32P-labelled aptamers and 0, 2, 20, 200 or 400 nM of non-labelled UTR 308 target RNA.…”

Therapeutic Targeting of HCV Internal Ribosomal Entry Site RNA

“…The most potent RNAs had K D values in the 50-100 nM range. These rather large inhibitor RNAs, of 60-75 nucleotides in length, interact with targets in the HCV IRES via loop-loop interactions in a fashion similar to those identified by Da Rocha Gomes et al [43], and by Romero-Lopez et al [36]. The consistent selection of such molecules would support a hypothesis that hairpin loop sequences are more accessible targets for therapeutics and do not have the disadvantage that intramolecular interactions within the target must be overcome, as would be the case if the duplex regions were targeted.…”

“…The aptamer recognition in both cases was mediated by looploop recognition motifs; in the HH363-50 molecule, an anchoring G:A pair was selected for that has been shown to be particularly advantageous not only for HCV-RNArecognizing aptamers, but also in the well-studied HIV Tar-Tar* complex [36,39,40]. To facilitate the IRES inhibition activity of the HCV-targeted aptamers, the loop-recognizing motif was subsequently fused with a hammerhead ribozyme motif, potentially adding catalytic cleavage activity to the RNA therapeutic.…”

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

“…Interestingly, inhibition of viral pathogenesis by engineered RNase P ribozyme has been shown before (Liu and Altman 1995;Bai et al 2008), suggesting that this methodology may be extended to other infectious agents. Nucleic acid-based interference provides promising therapeutic agents against many diseases caused by RNA viruses (de los Santos et al 2005;Lim et al 2008;Roy et al 2008;Romero-Lopez et al 2009). However, these tools show limitations regarding targeting efficiency, sequence specificity, as well as emergence of virus escape variants (Puerta-Fernandez et al 2003;Gitlin et al 2005;Schubert et al 2005;Thompson and Patel 2009).…”