Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures

Figures

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  Figure 1. CD spectra of G-rich RNA oligonucleotides with or without a 3′ overhang (tail) indicate the formation of parallel G4 structures in 100 mM KCl.A, diagram of an FAM-labeled G-quadruplex structure lacking (left) or containing (right) a 3′ overhang. Panels (B), (C), and (D) present the CD spectra of three different G-rich RNA oligonucleotides with (bottom) or without (top) a 3′ overhang. FAM, Fluorescein Amidite; G4, G-quadruplex.

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  Figure 2. NS3 binds tightly to G4-forming oligonucleotides with or without a 3′ overhang.A, the schematic representations of the three types of oligonucleotides, single-stranded RNA (ssRNA; positive control), tailless quadruplex, and 3′ tailed G4 structures. B, fluorescence anisotropy of increasing concentrations of NS3 with tailless G4s, HCVG4 (red circles), NONHCVG4 (blue circles), or NEGG4 (black circles). C, the fluorescence anisotropy measurements of NS3 with the 3′ tailed G4s, HCVG4-A20 (6 nM; red circles), NONHCVG4-A20 (6 nM; blue circles), NEGG4-A20 (5.4 nM; black circles). Anisotropy of the ssRNA is shown in green (B and C). The dissociation constants (Kd) were determined by fitting the fluorescence anisotropy data to the quadratic equation, for each of the oligonucleotides listed in (D). The fluorescence anisotropy data presented in panels (B) and (C) represent the average of the values from at least three independent binding experiments. Error bars depict the SD. HCV, hepatitis C virus; G4, G-quadruplex.

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  Figure 3. NS3 is active in a buffer that supports G4 folding.A, to test the effect of 100 mM KCl, the salt concentration at which G4 unfolding was studied, on the activity of the purified NS3, we performed an enzyme-coupled spectrophotometric ATPase assay which couples NADH oxidation with ATP hydrolysis. B, the assay was carried out in a reaction buffer containing 50 mM KCl (B; orange) or 100 mM KCl (B; green). ATP hydrolysis by NS3 (50 nM) was initiated with 5 μM, 100 μM, or 250 μM Poly (U). Experiments were performed in triplicate. Error bars depict SD. The difference between two groups is not significant (ns) when p > 0.05. p values were calculated from independent-samples t-tests. G4, G-quadruplex.

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  Figure 4. Determining saturating enzyme concentration for the G4 reporter duplex unwinding reaction. The reaction setup for the G4 reporter duplex unwinding reaction is shown in (A). The enzyme was preincubated with the G4 reporter substrate before addition of ATP. The reaction was quenched at increasing time points in a buffer containing 100 mM EDTA and 1% SDS. In this reaction, the unfolding of the G4 structure precedes the unwinding of the duplex region (B). A trapping strand is added to deter the reannealing of the unwound substrate (B). ∗ symbol in (B) indicates that the substrate is radiolabeled. C, in an effort to define the enzyme concentration that saturates the G4 reporter duplex substrate, we measured the amount of product formed with increasing concentrations of NS3 under multi-turnover conditions. The product formed with 400 nM, 600 nM, 800 nM, and 1000 nM NS3 concentrations are represented in green, blue (C) or cyan squares (D), black, and red colors, respectively (C and D). The amount of product measured at 10 min for each NS3 concentrations is presented in (D). Experiments were performed in triplicate. Error bars depict SD. The difference between two groups is statically…

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  Figure 5. NS3 unfolds the G4 structure within HCVG4-A20reporter duplex substrate; however, its activity is significantly reduced in the presence of a G4-stabilizing ligand.A, the standard reaction condition (green) involved the following factors: 2 nM HCVG4-A20 reporter duplex, 600 nM NS3, and 5 mM ATP. For the + PDS (red), -ATP (blue), and –NS3 (black) unwinding reactions, the same factors as the standard reaction were involved except that 0.25 μM PDS was added, or 5 mM ATP was left out, or the reaction was carried out in the absence of NS3, respectively (B and C). The ratio of product to substrate for each reaction time is plotted (C). The data for the standard reaction were fit to a double exponential equation with rate constant values of k1 =1.29 ± 0.28 min−1; k2 = 0.16 ± 0.04 min−1. Data from the experiment including PDS were best fit to a single exponential equation with a rate constant value of k = 0.18 ± 0.03 min−1. Experiments were executed in triplicate. Error bars depict SD. HCV, hepatitis C virus; G4, G-quadruplex; PDS, Pyridostatin.

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  Figure 6. NS3 unfolding of HCVG4 is affected by reaction conditions.A, the substrate and product from NS3 unfolding HCVG4 then unwinding the reporter duplex under multi-turnover or single-turnover conditions in 100 mM KCl are separated by PAGE. The gel image was quantified and the ratio of product to substrate for a given time point was plotted against time (B), with green and orange circles representing the data points for multi-turnover and single-turnover conditions, respectively. The gel image of HCVG4-A20 reporter duplex unwinding reaction in 100 mM KCl or LiCl is shown in (C), and the quantification of the gel image is presented in graph (D), with green and pink circles, respectively. Conditions in the absence of ATP (blue circles) or NS3 (black circles) were performed under the same conditions as the 100 mM LiCl reaction except that ATP or NS3 was omitted from the reaction (D). The data for multi-turnover (A and B) and 100 mM KCl (C and D) are re-represented from Figure 5 for comparison. The gel images for multi-turnover (A) and 100 mM KCl (C) are the same as the gel image shown in Figure 5B for the standard reaction. Experiments were performed in triplicate. Error bars represent…

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  Figure 7. HCVG4 unfolding activity of NS3 is directly proportional to the length of the 3′ overhang. NS3-dependent unwinding of HCVG4 reporter duplex substrates with 20, 9, or 2 nucleotides of single-stranded RNA on the 3′ overhang was carried out, and the fraction product formed was calculated and presented with green, blue, and red circles, respectively. The data for HCVG4-A20 are replotted from Figure 5 for comparison. The experiments were conducted in triplicate, and error bars depict SD. HCV, hepatitis C virus; G4, G-quadruplex.

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  Figure 8. G-quadruplex is a strong deterrent against the helicase activity of NS3.A, the G4-forming region within the HCVG4-A20 reporter duplex was mutated to create a control duplex (MUTHCVG4-A20) of the same length but unable to fold into a G4 structure. The amount of product formed at increasing time points from the control substrate in the presence of NS3 under multi-turnover (green) or single-turnover (orange) conditions and a reaction that contained T50 protein trap but no enzyme (black) are shown. B, under multi-turnover (green) or single cycle (orange) conditions, NS3 had significantly higher activity on MUTHCVG4-A20 than on HCVG4-A20. C, no significant difference in product formation was observed when NS3 unwound MUTHCVG4-A20 in a reaction buffer containing KCl (green) or LiCl (pink). D, in contrast, the product obtained from HCVG4-A20 was significantly lower in 100 mM KCl (green) than in 100 mM LiCl (pink). Each of the reactions were conducted under multi-turnover conditions unless otherwise specified. The data for HCVG4-A20 in (B and D) are replotted from Figure 6 for comparison. Each of the reactions were performed in triplicate. Error bars depict SD. The difference between two…

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  Figure 9. Both oligonucleotide sequence and the type of monovalent cation present in the reaction buffer affect NS3 G4RNA unfolding. Data from NS3-catalyzed unwinding of three G4 substrates with a reporter duplex (NEGG4-A20, NONHCVG4-A20, and HCVG4-A20 reporter duplexes) are presented. The reactions were carried out in a buffer containing 100 mM KCl (A) or LiCl (B). Data points for the reactions containing NS3 are depicted on graphs with black, blue, or red solid circles representing NEGG4-A20, NONHCVG4-A20, or HCVG4-A20, respectively (A and B). Unwinding reactions in the absence of NS3 are shown in black solid square, blue solid diamond, or red open circles representing NEGG4-A20, NONHCVG4-A20, or HCVG4-A20, respectively (A). The data for HCVG4-A20 (100 mM KCl) are replotted from Figure 5 for comparison. The data for HCVG4-A20 (100 mM LiCl) are replotted from Figure 6 for comparison. Experiments were performed in triplicate and error bars represent SD. HCV, hepatitis C virus; G4, G-quadruplex; G4RNA, G-quadruplex RNA.

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  Figure 10. Trapping assay allows direct measurement of G4RNA unfolding by NS3. A schematic representation of NS3-catalyzed G4RNA unfolding reaction using a trapping strand is shown in (A) and the reaction setup is presented in (B). The ability of NS3 to unfold 25 nM of FAM-labeled (indicated by F) HCVG4-A20, NEGG4-A20, or NONHCVG4-A20 was assessed. NS3 was incubated with the substrates. Upon addition of ATP and Mg+2, the unfolded G4 sequences were trapped by the Q-trap. The reaction was quenched in a buffer containing EDTA, SDS, and C-trap. The C-trap formed a duplex with excess Q-trap to prevent additional unfolding of the substrate after the reaction ended. C, the product formed from NEGG4-A20, NONHCVG4-A20, and HCVG4-A20 unfolding in the presence of NS3 is shown in solid black, blue, and red circles, respectively. For the reaction with trap alone (no NS3), the products formed from NEGG4-A20, NONHCVG4-A20, and HCVG4-A20 are shown in grayopen circles, diamonds, and squares, respectively. D, the product formed at 20 min from each of the substrates with (gray) or without (pink) NS3 is shown. The data for HCVG4-A20 in the presence of NS3 (C and D) are replotted from Fig. S7 for comparison.…

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  Figure 11. G4RNA-unfolding activity of NS3 is completely inhibited by intramolecular G-quadruplex.A, FAM-labeled NONHCVG4-A20 formed both intramolecular G4 and intermolecular G4 (higher order structure) in the presence of 100 mM KCl. The native polyacrylamide gel image shown in (A) displays the product formed from a trapping assay conducted in the presence (left) or absence (right) of NS3. The quantity of intramolecular or intermolecular G4 in the presence (B) or absence (C) of NS3 over time is presented in line graph. The trapped product data shown in panel (B) or (C) are replotted from Figure 10 for comparison. The data points for the trapped product in (B) are fit to a double exponential equation and the k1= 9.5 ± 2.8 min−1 and k2= 0.14 ± 0.0098 min−1. The intermolecular G4 data points (B) are fit to a single exponential decay and the k = 0.17 ± 0.044 min−1. The data points for the trapped product in the absence of NS3 (C) are fit to a single exponential equation and k = 0.14 ± 0.020 min−1. The scatter plots in (D and E) exhibits the amount of intramolecular (D) or intermolecular (E) G4 unfolded in the presence or absence of NS3 at 0 (gray circles for both panel D and E)or 20 min (red…