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. 2017 Dec 14;92(1):e01253-17.
doi: 10.1128/JVI.01253-17. Print 2018 Jan 1.

NS3 From Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

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Free PMC article

NS3 From Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

Ting Zhou et al. J Virol. .
Free PMC article

Abstract

Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1.IMPORTANCE Genotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important mechanical component possesses biochemical and structural properties that differ between common strains such as those of genotype 1b and a strain of HCV that replicates with exceptional efficiency (JFH-1, classified as genotype 2a). Indeed, unlike the inefficient genotype 1b NS3, which has been well studied, JFH-1 NS3 is a superhelicase with strong RNA affinity and high unwinding efficiency on a broad range of targets. Crystallographic analysis reveals architectural features that promote enhanced biochemical activity of JFH-1 NS3. These findings show that even within a single family of viruses, drift in sequence can result in the acquisition of radically new functional properties that enhance viral fitness.

Keywords: crystal structure; enzymology; evolution; hepatitis C virus; viral replication.

Figures

FIG 1
FIG 1
RNA binding affinities of JFH1-NS3 and gt1b-NS3 to RNAs with various overhang lengths. JFH1-NS3 and gt1b-NS3 variants are indicated with red circles and blue squares, respectively. RNA affinity was measured by filter binding, as described in Materials and Methods. (A) Affinity of JFH-1 NS3-4A (8.36 ± 0.29 nM) and gt1b NS3-4A (7.56 ± 0.33 nM) for single-stranded RNA (ssT34). (B) Affinity of JFH1-NS3 (6.56 ± 0.23 nM) and gt1b-NS3 (7.25 ± 0.17 nM) for a 12-base-pair duplex RNA with an 18-nt single-stranded 3′ overhang. (C) Affinity of JFH1-NS3 (7.69 ± 0.21 nM) and gt1b-NS3 (15.7 ± 0.61 nM) for a 12-base-pair duplex RNA with a 12-nt 3′ overhang. (D) Affinity of JFH1-NS3 (14.9 ± 0.52 nM) and gt1b-NS3 (60.4 ± 4.38 nM) for a 12-bp duplex RNA with a 6-nt 3′ overhang. The experiments were performed in triplicate, and error bars represent standard errors of the mean.
FIG 2
FIG 2
Association rate constants (kon) for JFH-1 NS3 and gt1b NS3 binding to RNAs with various overhang lengths. Rate constants were measured in a stopped-flow apparatus, as described in Materials and Methods. Data for JFH-1 NS3 and gt1b NS3 are indicated with red circles and blue squares, respectively. (A) The kon values for JFH-1 NS3 and gt1b NS3 on a 12-bp duplex RNA with an 18-nt 3′ overhang are 59 ± 4.6 μM−1 s−1 and 13 ± 2.0 μM−1 s−1, respectively. (B) The kon values for JFH-1 NS3 and gt1b NS3 on a 12-bp duplex RNA with a 12-nt 3′ overhang are 69 ± 6.4 μM−1 s−1 and 9.9 ± 3.3 μM−1 s−1, respectively. (C) The kon values for JFH-1 NS3 and gt1b NS3 on a 12-bp duplex RNA with a 6-nt 3′ overhang are 15 ± 1.9 μM−1 s−1 and 3.5 ± 1.1 μM−1 s−1, respectively. The experiments were performed in triplicate, and error bars represent standard errors of the mean.
FIG 3
FIG 3
Rate constants for functional NS3-RNA complex formation. JFH1-NS3, gt1b-NS3, and gt1b-NS3-ΔC7 are indicated by red circles, blue squares, and black diamonds, respectively. (A) Functional complex formation between NS3 variants and a 12-bp duplex RNA with a 6-bp 3′ overhang, resulting in rate constants of k1 = 0.37 ± 0.08 min−1 and k2 = 0.61 ± 0.15 min−1 for JFH-1 NS3, k1 = 0.09 ± 0.01 min−1 and k2 = 0.36 ± 0.11 min−1 for gt1b-NS3, and k2 = 0.33 ± 0.03 min−1 for gt1b-NS3-ΔC7, as described in Materials and Methods. (B) Functional complex formation between NS3 variants and an 18-bp duplex RNA with a 6-bp 3′ overhang, resulting in rate constants of k1 = 0.23 ± 0.02 min−1 and k2 = 0.48 ± 0.11 min−1 for JFH1-NS3 and k1 = 0.11 ± 0.04 min−1 and k2 = 0.26 ± 0.06 min−1 for gt1b-NS3. (C) Rapid functional complex formation by JFH-1 NS3 in the absence of trap RNA (ssT34) on a 12-bp duplex RNA flanked by a 6-bp 3′ overhang. JFH1-NS3 completely unwinds the duplex extremely rapidly, with a rate constant that is higher than can be computed for this hand-mixed experiment (>1.4 min−1, which should be considered a lower bound on the rate constant), whereas gt1b-NS3 unwinds at a time scale similar to that for single-cycle conditions (0.09 ± 0.01 min−1). (D) Functional complex formation between NS3 variants and a 12-bp duplex RNA with a 3-nt 3′ overhang, resulting in rate constants of 300 ± 54 × 10−4 min−1 and 5.7 ± 0.49 × 10−4 min−1, for JFH-1 NS3 and gt1b NS3, respectively. Experiments were performed in triplicate, and error bars represent standard errors of the mean.
FIG 4
FIG 4
Single-cycle RNA unwinding efficiencies of JFH-1 NS3 and gt1b NS3 (A) The rate constants (kunw values) and amplitudes (in parentheses) for unwinding a 12-bp RNA duplex with a 6-nt 3′ overhang are 1.8 ± 0.15 min−1 (0.69 ± 0.02) and 1.9 ± 0.11 min−1 (0.50 ± 0.01) for JFH-1 NS3 and gt1b NS3, respectively. (B) The rate constants and amplitudes for unwinding an 18-bp duplex RNA with a 6-nt 3′ overhang are 1.6 ± 0.17 min−1 (0.41 ± 0.01) and 1.7 ± 0.18 min−1 (0.24 ± 0.01) for JFH-1 NS3 and gt1b NS3, respectively. (C) The rate constants and amplitudes for unwinding an 18-mer duplex RNA with a 12-nt 3′ overhang are 2.5 ± 0.14 min−1 (0.69 ± 0.01) and 2.5 ± 0.22 min−1 (0.41 ± 0.01) for JFH-1 NS3 and gt1b NS3, respectively. Experiments were performed in triplicate, and error bars represent standard errors of the mean.
FIG 5
FIG 5
Structural comparison of JFH-1 NS3 and gt1b NS3 (PDB 3o8B). (A) Alignment of JFH-1 NS3 (green) and gt1b NS3 (yellow) in different views shows that the positional shift of D2 is caused by a 9.6° twist in the β14/15 hairpin (framed with a red rectangle). The RNA entry site is indicated with a blue arrow. (B) In the JFH-1 NS3 structure (green), the conserved RNA binding motifs IV (residues 365 to 372) and V (residues 411 to 419) shift outwards by about 6.8 Å and 4.2 Å, respectively, resulting in a longer RNA binding groove than in gt1b NS3 (yellow). (C) Comparison of the surfaces of JFH-1 NS3 (left panel) and gt1b NS3 (right panel). D2 is shown in gray for both variants. The gt1b NS3 variant contains an H-bond network (shown in red) that is absent in JFH-1 NS3, and the corresponding parts in both structures are indicated with a black square. (D) The H-bond network between D3 and D2 of gt1b NS3; a side chain atom of Asp555 and a main chain atom of Gly554 interact with side chain atoms of Arg393.
FIG 6
FIG 6
Comparison of the surface electrostatic potentials of JFH-1 NS3 (A) and gt1b NS3 (PDB 3o8B) (B). Left panels, RNA entry sites are indicated with yellow arrows. The surface of D2 has a smaller negatively charged patch adjacent to the RNA entry site in JFH-1 NS3 than in gt1b NS3, due to the twist of hairpin β14/15. Right panels, RNA entry sites are indicated with yellow dots. Both JFH-1 and gt1b NS3 contain a negatively charged pocket (yellow ellipse) near the RNA entry site. However, the rotation of D2 in JFH-1 NS3 causes this patch to be buried, resulting in a smaller presentation of negative charge. This decrease in surface-exposed negative charge may facilitate RNA attraction and better define RNA orientation upon binding to the protein.
FIG 7
FIG 7
Comparison of the NS3 sequences from genotype 1 and 2 HCV strains. (A) Amino acid variations between JFH-1 and gt1b NS3-helicase domains are mapped on the JFH-1 NS3 structure (shown in red). (B) Amino acid variations between JFH-1 and other gt2a NS3-helicase domains are mapped on the JFH-1 NS3 structure (shown in red). The minor differences, such as I/L/V, S/T, or G/A substitutions, are not mapped in panel A or B. The statistics of the variations in both panels A and B are shown in Table S1 in the supplemental material. (C) NS3 sequences (801) from HCV genotypes 1 and 2 were analyzed by principal-component analysis, with the top three components graphed on x, y, and z axes. (D) NS3 sequences (123) from HCV genotype 2 were analyzed by principal-component analysis as for panel C.

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