Inhibition of hepatitis C virus (HCV) RNA polymerase by DNA aptamers: mechanism of inhibition of in vitro RNA synthesis and effect on HCV-infected cells

Abstract

We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

FIG. 1.

Effects of ODNs and ORNs on RdRp activity of NS5B. RdRp assays were performed as described in Materials and Methods. Strain H77 NS5B at 150 nM was incubated in a 20-μl RdRp reaction mixture with 86 nM (−)IRES RNA and increasing amounts of 81-nt ODNs (ODN 27 or 127) or 35-nt ODNs (ODN 27v or 127v). (A) ODN 27 (open circles) and ODN 27v (closed circles); (B) ODN 127 (open circles) and ODN 127v (closed circles). The amount of RNA synthesis was determined by measuring the TCA-precipitable radioactivity. The results, which correspond to the mean values of three independent experiments, are expressed as relative activity, in which 100% activity corresponds to the enzymatic activity in the absence of ODN aptamers. (C) The RdRp assay was performed in the presence of [α-³²P]UTP without an ODN (lane 1) or with 100 nM, 200 nM, 400 nM, 800 nM, 2 μM, and 4 μM ODN 27v (lanes 2 to 7, respectively) or ODN 127v (lanes 8 to 13, respectively). The ³²P-labeled reaction products were denatured and loaded onto a 6% denaturing polyacrylamide gel. Lane −, incubation without enzyme; lane M, RNA size marker. (D) Increasing concentrations of DNA aptamers 27v (closed circles) and 127v (closed triangles) or oligonucleotides corresponding to the RNA version of aptamers 27v (open circles) and 127v (open triangles) were added to the polymerase reaction mixture. Error bars represent the standard deviations.

FIG. 2.

Effects of ODNs 27v and 127v on the activities of different viral polymerases. Polymerase assays were performed as described in Materials and Methods. (A and B) Effects of ODN 27v (A) and ODN 127v (B) on the activities of the HIV-1 RT (open squares), HCV H77-NS5B (closed circles), poliovirus 3D^pol (closed triangles), and GBV-B NS5B (open triangles); (C) effects of ODN 27v (circles) and ODN 127v (triangles) on the activities of genotype 1a (closed symbols) and genotype 2a (open symbols) NS5B. The results correspond to the mean values of four independent experiments. They are expressed as relative activity, with 100% activity corresponding to the enzymatic activity in the absence of ODN. Error bars represent the standard deviations.

FIG. 3.

Determination of parameters of binding of aptamers 27v and 127v to HCV NS5B. The 5′-end-labeled ODN 27v (A) and ODN 127v (B) were incubated in 10 μl of selection buffer with increasing amounts of H77-NS5B (31.2 nM to 1 μM). After incubation, the NS5B-ODN complexes were separated from the free ODNs by electrophoresis on a nondenaturing polyacrylamide gel (left panels). The percentage of bound ODNs (right panels) was determined as described in Materials and Methods. The results correspond to the mean values of four independent experiments. Error bars represent the standard deviations.

FIG. 4.

Gel shift competition assay with HCV NS5B. (A) H77-NS5B (500 nM) incubated in 10 μl RdRp reaction mixture with 10,000 cpm ³²P-labeled (−)IRES RNA (13 nM) and increasing amounts of ODN 27v (0.5 μM, 1 μM, 2 μM, 5 μM, and 10 μM [lanes 1 to 5, respectively]) or ODN 127v (0.5 μM, 1 μM, 2 μM, 5 μM, and 10 μM [lanes 6 to 10, respectively]) After incubation, the complexes were separated from free RNA by electrophoresis on a nondenaturing polyacrylamide gel. Lane −, no NS5B; lane +, no ODN plus NS5B. (B) The labeled bands corresponding to free RNA were quantified, and the percentage was plotted against the concentration of the DNA aptamers (closed circles, ODN 27v; open circles, ODN 127v). The data correspond to the mean values of three independent experiments. Error bars represent the standard deviations.

FIG. 5.

RdRp activity in a single-cycle round of synthesis. (A and B) Inhibition curves for ODN 27v (A) and ODN 127v (B) under single-cycle conditions. H77-NS5B (150 nM), (−)IRES template (86 nM), and CTP and GTP (0.5 mM each) were preincubated for 30 min at 30°C. Heparin was added, and elongation was started by the addition of 0.5 mM ATP plus [α-³²P]UTP. Various concentrations of ODN were added at the same time as RNA and NS5B (closed circles) or as ATP and [α-³²P]UTP at the beginning of elongation (closed triangles). In a third experiment (open circles), ODN was added after the formation of the RNA-NS5B complex and the mixture was incubated for 30 min at 30°C before the addition of heparin and ATP plus [α-³²P]UTP. The data correspond to the means of four independent experiments. Error bars represent the standard deviations.

FIG. 6.

Analysis of ODN 27v and 127v inhibition in gel-based initiation and elongation assays. (A) Sequence of the 23-nt RNA template used in gel-based initiation and elongation assay. (B) Inhibition of initiation step; 1.13 μM H77-NS5B was incubated for 30 min with 1 μM 23-nt RNA template, [³²P]ATP, and GTP without aptamer (lanes 5 and 10) or with 0.2 μM, 1 μM, 5 μM, or 25 μM ODN 127v (lanes 1 to 4, respectively) or ODN 27v (lanes 6 to 9, respectively). (C and D) Analysis of the initiation products synthesized without RNA template with [³²P]ATP and GTP (C) or with [³²P]ATP, CTP, and GTP (D). The lanes are as described above for panel B. (E) Inhibition of the elongation step; 1.13 μM H77-NS5B was incubated for 30 min with 1 μM 23-nt RNA template, ATP, and GTP without aptamer. Elongation was started by addition of UTP, CTP, [³²P]ATP, and aptamers. The lanes corresponding to the elongation reaction with aptamer dilutions or without aptamers are as described above for panel B.

FIG. 7.

UV melting curves for the aptamers. The thermal denaturation of ODNs 27v (A) and ODN 127v (B) was monitored at 260 nm, as described in Materials and Methods. Denaturation and renaturation curves are shown by closed and open circles, respectively, and their corresponding first derivatives are indicated by continuous and dotted lines. OD, optical density. (C and D) Secondary structures of ODN 27v (C) and ODN 127v (D) predicted by mfold software.

FIG. 8.

(A) Inhibition of JFH1 virus replication. The culture supernatant of Huh7-QR cells producing JFH1 virus (5 × 10⁴ infectious viruses/500 μl) was used to infect naïve Huh7-QR cells. Viral particles were added either alone; with interferon at 150 U·ml⁻¹; or with the various ODNs at 100 nM, 1 μM, or 5 μM. After a 48-h incubation, cellular RNAs were extracted from infected cells and the viral RNA copies were quantified by real-time RT-PCR. The results are expressed as a percentage of those obtained with cells infected with the virus alone. They correspond to the means of three independent infection experiments. Error bars represent the standard deviations. (B) Titration of HCV particles produced in the presence of DNA aptamers. A total of 5 × 10⁴ infectious viruses/250 μl was used to infect naïve Huh7-QR cells in the presence or the absence of the DNA aptamers. After a 48-h incubation, the supernatants were used to infect Huh7-QR cells. HCV-infected cells were revealed after 48 h of culture with an anti-E2 antibody and Alexa Fluor 594-conjugated secondary antibodies, as described in Materials and Methods. The results are expressed as a percentage of those obtained with virus produced in the absence of aptamers. They correspond to the means of three independent infection experiments. Error bars represent the standard deviations. The left inset shows a typical focus of infected cells observed under fluorescent light. The right inset corresponds to the field observed under a phase-contrast microscope.