Research ArticleCHEMICAL BIOLOGY

A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti–hepatitis C target

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Science Advances  01 Apr 2016:
Vol. 2, no. 4, e1501535
DOI: 10.1126/sciadv.1501535
  • Fig. 1 Graphical representations of G-rich sequences consensus in the HCV genome.

    (A and B) A total of 1056 partial cds of the C gene for (A)subtype 1a and 1025 partial cds of the C gene for (B) subtype 1b were retrieved from the National Center for Biotechnology Information Web site (www.ncbi.nlm.nih.gov) and the HCV database (www.hcv.lanl.gov/) and aligned using WebLogo software.

  • Fig. 2 Synthetic HCV G-rich sequences form G4 RNAs.

    (A) Formation of compact G4 RNAs characterized on the basis of species that move more rapidly than the G4-mutated oligonucleotides. Lane 1, RNA1a–Mut-F; lane 2, RNA1a-FAM; lane 3, RNA1b-FAM; lane 4, RNA1b–Mut-F. (B) G4 structures of RNA1a evidenced by 1H NMR. The chemical shift of Hoogsteen imino peaks in the range of 10.0 to 11.5 ppm was partially suppressed through AS-RNA1a (100 μM) in favor of Watson-Crick imino peaks. The expanded imino proton signals were analyzed by TopSpin 2.0 software. (C) G4 structures of RNA1b evidenced using 1H NMR.

  • Fig. 3 G4 DNA ligand can stabilize target HCV G4 RNAs.

    (A) The structure of compound PDP. (B and C) Melting profiles of RNA1a (8.0 μM) or RNA1b (8.0 μM) were recorded in 10 mM tri-HCl buffer (pH 7.0) (100 mM KCl), in the absence or presence of PDP (8.0 μM).

  • Fig. 4 G4 ligands inhibit RNA-dependent RNA synthesis and HCV C gene expression through G4 RNA stabilization.

    (A) Schematic representation of the RNA stop assay. The artificial RNA template containing HCV G-rich or G4-mutated sequences was used. (B) The extended RNAs of template 1a-G4 (lanes 2 to 7) or template 1a-G4 mut (lanes 8 to 11) analyzed through denaturing PAGE. The arrows indicate the positions of the full-length product, G4-pausing product, and free primer. The fully extended product and the template RNA formed a stable duplex, which was not denatured and moved much slower than the corresponding ssRNA. Lane 1, RNA markers (p15, m17, m19, m21, and m39-1a in table S4); lanes 2 and 3, no enzyme or no PDP control; lanes 4 to 7, G4 ligand–dependent inhibition; lanes 8 and 9, no enzyme or no PDP control; lanes 10 and 11, treatment with the G4 ligand. (C) Western blot analysis showing the suppression of HCV C gene expression through G4 ligands. The values indicate the percentage of densitometry of the HCV Core protein relative to β-actin. DMSO, dimethyl sulfoxide; nt, nucleotides.

  • Fig. 5 G4 ligands suppress intracellular HCV replication.

    (A) RT-qPCR was used to determine the amount of HCV RNA in HCV Con1/JFH1-infected Huh-7.5.1 cells treated in triplicate with the G4 ligands or control (DMSO or IFN-α). IFN-α was used at 150 ng/ml. The values observed were normalized to GAPDH. All data are presented as the means ± SEM from three independent experiments. The error bars reflect the SD. G4 ligand groups versus DMSO group, *P < 0.05. The primers were designed to target the C gene of Con1/JFH1 RNA. (B) RT-qPCR was performed, and the primers were designed to target the 5′UTR of Con1/JFH1 RNA. (C) Western blot analysis showed the suppression of intracellular HCV replication. A commercial anti–HCV Core 1b antibody was used, and the values indicate the percentage of densitometry of the target HCV protein relative to β-actin. (D) Western blot analysis was performed, and a commercial anti–HCV nonstructural protein 3 (NS3) antibody was used for detection.

  • Fig. 6 G4-disruptive mutations in the HCV C gene inhibit G4 ligand–virus interactions.

    (A) RT-qPCR was performed. The primers were designed to target the C gene of J6/JFH1 virus. All data are presented as the means ± SEM from three independent experiments. The error bars reflect the SD. (B) Western blot analysis was performed. The values indicate the percentage of densitometry of the target HCV NS3 protein relative to β-actin. Lane 1, no HCV control; lanes 2 to 7, J6/JFH1–G4-Mut virus; lanes 8 to 13, J6/JFH1 virus.

  • Table 1 Sequences of some oligomers used in our studies.
    OligomerSequence (from 5′ to 3′)
    RNA1a5′-GGGCUGCGGGUGGGCGGGA-3′
    RNA1b5′-GGGCAUGGGGUGGGCAGGA-3′
    RNA1a-FAM5′-FAM-AGGGCUGCGGGUGGGCGGGA-3′
    RNA1a–Mut-F5′-FAM-AGAGCUGCGAGUGAGCGAGA-3′
    RNA1b-FAM5′-FAM-AGGGCAUGGGGUGGGCAGGA-3′
    RNA1b–Mut-F5′-FAM-AGAGCAUGGAGUGAGCAAGA-3′
    AS-RNA1a5′-UCCCGCCCACCCGCAGCCC-3′
    AS-RNA1b5′-UCCUGCCCACCCCAUGCCC-3′

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/4/e1501535/DC1

    Materials and Methods

    Fig. S1. Structural illustration of a G-quartet.

    Fig. S2. Conservation analysis of HCV genomes.

    Fig. S3. Premade sequence alignment in the central part of the HCV C gene, between positions +253 and +294.

    Fig. S4. Premade sequence alignment in the central part of the HCV C gene, between positions +253 and +294.

    Fig. S5. Premade sequence alignment in the central part of the HCV C gene, between positions +253 and +294.

    Fig. S6. Expansion of the 1H NMR spectra of RNA1a.

    Fig. S7. Expansion of the 1H NMR spectra of RNA1b.

    Fig. S8. Prediction of the RNA secondary structure of the C gene (subtype 1a) using free-energy minimization.

    Fig. S9. Prediction of the RNA secondary structure of the C gene (subtype 1b) using free-energy minimization.

    Fig. S10. G4 formation in a long structural context evidenced by 1H NMR.

    Fig. S11. Synthetic HCV G-rich sequences form parallel G4 RNAs.

    Fig. S12. G4 structure of RNA1a is more stable than that of RNA1b.

    Fig. S13. G4 RNAs are characterized in the presence of alkali metal ions (K+, Na+, or Li+).

    Fig. S14. HCV G4 RNA structures are destabilized through the ASO.

    Fig. S15. CD melting curves of HCV G-rich RNAs.

    Fig. S16. Influence of different alkali metal ions on the thermal stabilities of HCV G4 RNAs.

    Fig. S17. Analysis of concentration-independent melting curves of target HCV RNAs.

    Fig. S18. CD melting studies of target HCV RNAs.

    Fig. S19. Structures of TMPyP4 and TMPyP2.

    Fig. S20. G4 ligand stabilizes target HCV G4 RNAs.

    Fig. S21. Little interaction is observed between the G4 ligand and G4-mutated RNAs.

    Fig. S22. Schematic depiction of the inhibition of FRET through the binding between PDP and G4 RNA.

    Fig. S23. PDP binds to target G4 RNA and inhibits the trap by the corresponding ASO.

    Fig. S24. G4 ligand inhibits RNA-dependent RNA synthesis through G4 RNA stabilization.

    Fig. S25. Map of the plasmid 24480 (pMO29) and a sequenced portion of this plasmid for verification.

    Fig. S26. TMPyP2 does not stabilize G4 RNA for RNA1b.

    Fig. S27. G4 ligands do not suppress the expression of the HCV C gene containing a G4-mutated sequence.

    Fig. S28. G4 ligands repress the in vitro expression of EGFP through G4 RNA stabilization.

    Fig. S29. G4 ligands do not repress the in vitro expression of EGFP in empty vector or G4-mutated plasmids.

    Fig. S30. Sequence of the C gene for HCV JFH1 virus.

    Fig. S31. Premade sequence alignment in the central part of the HCV C gene (subtype 2a), between positions +253 and +296.

    Fig. S32. Premade sequence alignment in the central part of the HCV C gene (subtype 2a), between positions +253 and +296.

    Fig. S33. Premade sequence alignment in the central part of the HCV C gene (subtype 2a), between positions +253 and +296.

    Fig. S34. Graphical representation of G-rich consensus sequences in genotype 2a HCV genomes.

    Fig. S35. G4 RNA structure of RNA2a evidenced in different studies.

    Fig. S36. G4 ligands inhibit intracellular HCV JFH1 replication.

    Fig. S37. Sequence of the C gene for HCV H77.

    Fig. S38. Sequence of the C gene for HCV Con1.

    Fig. S39. G4 ligands suppress intracellular HCV H77/JFH1 replication.

    Fig. S40. Western blot analysis shows suppression of intracellular HCV H77/JFH1 replication through G4 ligands.

    Fig. S41. Detection of HCV RNA using Tth-based RT-qPCR.

    Fig. S42. Structure of biotin-PDP.

    Fig. S43. Biotin modification on PDP does not impair the stabilization of HCV G4 RNAs.

    Fig. S44. Pull down of G4 RNA through biotin-PDP.

    Fig. S45. HCV G4 RNAs evidenced by a selective G4 affinity probe.

    Fig. S46. G4 ligands do not inhibit influenza A virus without a G-rich region.

    Fig. S47. ASOs destabilize the HCV G4s and show stimulatory effects on viral replication.

    Fig. S48. Target HCV sequences form intracellular G4 structures.

    Fig. S49. Target HCV sequences form intracellular G4 structures probed by the G4 ligand.

    Fig. S50. G4 ligand binds to the target G4 site in the full HCV genome under physiological conditions.

    Table S1. GenBank accession numbers of the HCV genomes analyzed.

    Table S2. List of 1056 sequenced partial cds of the C genes for subtype 1a.

    Table S3. List of 1025 sequenced partial cds of the C genes for subtype 1b.

    Table S4. Sequences of oligomers used in our studies.

    Table S5. Calculated Tm of synthetic HCV G-rich sequences in the presence of different alkali metal ions.

    Table S6. List of 143 sequenced partial cds of the C genes for subtype 2a.

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Structural illustration of a G-quartet.
    • Fig. S2. Conservation analysis of HCV genomes.
    • Fig. S3. Premade sequence alignment in the central part of the HCV C gene, between positions +253 and +294.
    • Fig. S4. Premade sequence alignment in the central part of the HCV C gene, between positions +253 and +294.
    • Fig. S5. Premade sequence alignment in the central part of the HCV C gene, between positions +253 and +294.
    • Fig. S6. Expansion of the 1H NMR spectra of RNA1a.
    • Fig. S7. Expansion of the 1H NMR spectra of RNA1b.
    • Fig. S8. Prediction of the RNA secondary structure of the C gene (subtype 1a) using free-energy minimization.
    • Fig. S9. Prediction of the RNA secondary structure of the C gene (subtype 1b) using free-energy minimization.
    • Fig. S10. G4 formation in a long structural context evidenced by 1H NMR.
    • Fig. S11. Synthetic HCV G-rich sequences form parallel G4 RNAs.
    • Fig. S12. G4 structure of RNA1a is more stable than that of RNA1b.
    • Fig. S13. G4 RNAs are characterized in the presence of alkali metal ions (K+, Na+, or Li+).
    • Fig. S14. HCV G4 RNA structures are destabilized through the ASO.
    • Fig. S15. CD melting curves of HCV G-rich RNAs.
    • Fig. S16. Influence of different alkali metal ions on the thermal stabilities of HCV G4 RNAs.
    • Fig. S17. Analysis of concentration-independent melting curves of target HCV RNAs.
    • Fig. S18. CD melting studies of target HCV RNAs.
    • Fig. S19. Structures of TMPyP4 and TMPyP2.
    • Fig. S20. G4 ligand stabilizes target HCV G4 RNAs.
    • Fig. S21. Little interaction is observed between the G4 ligand and G4-mutated RNAs.
    • Fig. S22. Schematic depiction of the inhibition of FRET through the binding between PDP and G4 RNA.
    • Fig. S23. PDP binds to target G4 RNA and inhibits the trap by the corresponding ASO.
    • Fig. S24. G4 ligand inhibits RNA-dependent RNA synthesis through G4 RNA stabilization.
    • Fig. S25. Map of the plasmid 24480 (pMO29) and a sequenced portion of this plasmid for verification.
    • Fig. S26. TMPyP2 does not stabilize G4 RNA for RNA1b.
    • Fig. S27. G4 ligands do not suppress the expression of the HCV C gene containing a G4-mutated sequence.
    • Fig. S28. G4 ligands repress the in vitro expression of EGFP through G4 RNA stabilization.
    • Fig. S29. G4 ligands do not repress the in vitro expression of EGFP in empty vector or G4-mutated plasmids.
    • Fig. S30. Sequence of the C gene for HCV JFH1 virus.
    • Fig. S31. Premade sequence alignment in the central part of the HCV C gene (subtype 2a), between positions +253 and +296.
    • Fig. S32. Premade sequence alignment in the central part of the HCV C gene (subtype 2a), between positions +253 and +296.
    • Fig. S33. Premade sequence alignment in the central part of the HCV C gene (subtype 2a), between positions +253 and +296.
    • Fig. S34. Graphical representation of G-rich consensus sequences in genotype 2a HCV genomes.
    • Fig. S35. G4 RNA structure of RNA2a evidenced in different studies.
    • Fig. S36. G4 ligands inhibit intracellular HCV JFH1 replication.
    • Fig. S37. Sequence of the C gene for HCV H77.
    • Fig. S38. Sequence of the C gene for HCV Con1.
    • Fig. S39. G4 ligands suppress intracellular HCV H77/JFH1 replication.
    • Fig. S40. Western blot analysis shows suppression of intracellular HCV H77/JFH1 replication through G4 ligands.
    • Fig. S41. Detection of HCV RNA using Tth-based RT-qPCR.
    • Fig. S42. Structure of biotin-PDP.
    • Fig. S43. Biotin modification on PDP does not impair the stabilization of HCV G4 RNAs.
    • Fig. S44. Pull down of G4 RNA through biotin-PDP.
    • Fig. S45. HCV G4 RNAs evidenced by a selective G4 affinity probe.
    • Fig. S46. G4 ligands do not inhibit influenza A virus without a G-rich region.
    • Fig. S47. ASOs destabilize the HCV G4s and show stimulatory effects on viral replication.
    • Fig. S48. Target HCV sequences form intracellular G4 structures.
    • Fig. S49. Target HCV sequences form intracellular G4 structures probed by the G4 ligand.
    • Fig. S50. G4 ligand binds to the target G4 site in the full HCV genome under physiological conditions.
    • Table S1. GenBank accession numbers of the HCV genomes analyzed.
    • Table S2. List of 1056 sequenced partial cds of the C genes for subtype 1a.
    • Table S3. List of 1025 sequenced partial cds of the C genes for subtype 1b.
    • Table S4. Sequences of oligomers used in our studies.
    • Table S5. Calculated Tm of synthetic HCV G-rich sequences in the presence of different alkali metal ions.
    • Table S6. List of 143 sequenced partial cds of the C genes for subtype 2a.

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