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.

Stem-loop SL2 is a self-interacting palindromic sequence that has been identified within the hepatitis C virus genome (HCV). While, RNA dimerization of the HCV genome has been observed in vitro with short RNA sequences, the role of a putative RNA dimerization during viral replication has not been elucidated. To determine the effect of genomic dimerization on viral replication, we introduced mutations into SL2 predicted to disrupt genomic dimerization. Using surface plasmon resonance, we show that mutations within the SL2 bulge impact dimerization in vitro. Transfection of Huh7 cells with luciferase-encoding full-length genomes containing SL2 mutations abolishes viral replication. Luciferase expression indicates that viral translation is not or slightly affected and that the viral RNA is properly encapsidated. However, RT-qPCR analysis demonstrates that viral RNA synthesis is drastically decreased. In vitro synthesis experiments using the viral recombinant polymerase show that modifications of intra-molecular interactions have no effect on RNA synthesis, while impairing inter-molecular interactions decreases polymerase activity. This confirms that dimeric templates are preferentially replicated by the viral polymerase. Altogether, these results indicate that the dimerization of the HCV genomic RNA is a crucial step for the viral life cycle especially for RNA replication. RNA dimerization could explain the existence of HCV recombinants in cell culture and patients reported recently in other studies.

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.