Binding of cellular nucleolin with the viral core RNA G-quadruplex structure suppresses HCV replication

Figure 1.. Six representative conserved PQS sites, CD and Tm melting curves. (A) Six conserved PQS (core/E1/NS3/NS4B/NS5A/NS5B) sites of the HCV 1a H77 genome. (B) Synthetic RNA PQS samples (8.0 μM) annealed in 10 mM Tris–HCl buffer (pH 7.0) containing 100 mM KCl were heated to 90°C and cooled to RT at a rate of 1°C min–1. The CD spectra were recorded at 25°C with a scan range from 320 to 200 nm. (C) The Tm thermal stability of each G4 RNAs was measured by recording the molar ellipticity at 264 nm as a function of temperature. Tm values for each oligonucleotide were calculated as described in Materials and Methods.

Figure 2.. Direct interaction and colocalization between HCV core RNA G4 and NCL in Huh7.5.1 cells. (A) RT-qPCR and Western blot analysis of HCV RNA and core protein expression. (B) G4 pull-down and Western blot. The fourth lane represents cell lysates directly used for western blot. β-actin was used as an internal control. (C) Colocalization of HCV core RNA G4 with NCL in Huh7.5.1 cells by confocal immunofluorescence. (D) Analysis of colocalization of wild-type HCV and modified G4 HCV with NCL in Huh7.5.1 cells by confocal immunofluorescence. Images in C and D with magnification 63×.

Figure 3.. The effects of G4 ligands on the colocalization of NCL and HCV core RNA G4 in Huh7.5.1 cells by confocal immunofluorescence analysis. Confocal immunofluorescence images with magnification 63×.

Figure 4.. NCL-R3–4C can stabilize target core RNA G4 and inhibit the trap by the corresponding ASO. (A) Schematic depiction of the inhibition of FRET. (B) Time course of the fluorescence intensity (F – F0) of a dual-labelled probe with increasing amounts of NCL-R3–4C. The final concentrations of HCV core RNA G4-dual and AS-HCV core RNA G4 were 200 nM and 2.0 mM, respectively. F – F0 = (real time fluorescence intensity – initial fluorescence intensity)/initial fluorescence intensity. (C) G4 structure of HCV core RNA G4 evidenced by 1H NMR. The final concentrations of HCV core RNA G4 and ASO were 0.5 and 0.1 mM, respectively.

Figure 5.. NCL is associated with HCV particles. Density gradient centrifugation analysis (A) and TCID50 (B). (C) Each purified HCV virion was assessed by Western blot. Jc1 virus was used as a parallel control with no flag tag.

Figure 6.. NCL suppresses HCV expression. RT-qPCR and western blot analysis of NCL expression after NCL overexpression or silencing. RT-qPCR (A) and western blot (B) analysis of NCL mRNA and protein expression after transfection of pc-NCL or p-sh-NCL in Huh7.5.1 cells or LO2 cells. pc-NCL vector group versus Huh7.5.1 cells or pcDNA3.1 vector group, **P < 0.01. p-sh-NCL vector group versus pSilencer1.0-U6 vector group, ***P < 0.001. (C) NCL suppresses HCV expression by confocal immunofluorescence analysis. Interferon-α (IFN-α, 150 ng/ml), a well-known effective cytokine for HCV treatment, was used as a positive control.

Figure 7.. NCL suppresses HCV replication by RT-qPCR analysis. (A, B) HCV infection promotes NCL mRNA (A) and protein expression levels (B) in Huh7.5.1 and HCVcc-Huh7.5.1 cells. *P < 0.05. (C) Overexpression of NCL inhibited HCV RNA replication. (D) Knockdown of NCL promoted HCV RNA replication. (E) Overexpression of NCL did not affect HCV-G4-Mut RNA replication. (F) Knockdown of NCL did not affect HCV-G4-Mut RNA replication. GADPH was used as the internal control.