Abstract
DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or “G4” that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3′end of the HCV (−) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy’ of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.
Highlights
DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or “G4” that are based on the stacking of G-quartets
We evaluated the ability of Phen-DC3 to interact with HCV110-131 G4 using the classical fluorescence resonance energy transfer (FRET) melting assay[32] with a 5′-FAM/3′-TAMRA labelled oligonucleotide (Fig. 2D,E)
RNA dependent RNA synthesis by Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) is affected by RNA G4. Because this RNA G4 is located at the 3′-end of the (−)strand, which is the initiation site for the (+)strand replication by the RdRp, we investigated the putative role of this structure on RNA synthesis by HCV polymerase in vitro
Summary
DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or “G4” that are based on the stacking of G-quartets. DNA and RNA guanine-rich regions can fold into non-canonical secondary structures called G-quadruplexes or “G4”. These structures result from the planar association of four guanines connected through hydrogen bonding. Its plus-strand RNA genome of 9600 nt is flanked by 5′ and 3′ untranslated regions (UTRs) that fold in several stem-loop structures engaged in short or long-range RNA-RNA tertiary interactions[16,17] These interactions are essential for viral replication[18,19,20]. We show that Phen-DC3, a known G-quadruplex ligand, inhibits HCV viral replication in cells As this region of the (−)strand RNA of HCV is highly conserved, targeting the G4 structure by ligands might be a new therapeutic pathway
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