Dynamic Interconversions of HCV Helicase Binding Modes on the Nucleic Acid Substrate

Abstract

The dynamics involved in the interaction between hepatitis C virus nonstructural protein 3 (NS3) C-terminal helicase and its nucleic acid substrate have been the subject of interest for some time given the key role of this enzyme in viral replication. Here, we employed fluorescence-based techniques and focused on events that precede the unwinding process. Both ensemble Förster resonance energy transfer (FRET) and ensemble protein induced fluorescence enhancement (PIFE) assays show binding on the 3' single-stranded overhang of model DNA substrates (>5 nucleotides) with no preference for the single-stranded/double-stranded (ss/ds) junction. Single-molecule PIFE experiments revealed three enhancement levels that correspond to three discrete binding sites at adjacent bases. The enzyme is able to transition between binding sites in both directions without dissociating from the nucleic acid. In contrast, the NS3 mutant W501A, which is unable to engage in stacking interactions with the DNA, is severely compromised in this switching activity. Altogether our data are consistent with a model for NS3 dynamics that favors ATP-independent random binding and sliding by one and two nucleotides along the overhang of the loading strand.

Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code

“…With the completely double-stranded Cy3-S19/X 19 mer, the resulting E pr was reduced to the residual value of ∼0.05, where this residual E pr was due to the bleed-through of the Cy3 signal into the Cy5 channel as we had observed previously. 28 The E pr for NS3h Y618AzF-Cy5 dropped in a distance-dependent manner as the length of the duplex on the substrate increased in length, consistent with what we had observed previously using a cysteine-Cy5 labeled NS3h construct (NS3h Cys-Cy5). 28 However, it should be noted that the FRET efficiencies for NS3h Y618AzF-Cy5 reported here in Figure 3B are about 2fold higher than those reported previously for the NS3h Cys-Cy5 construct with a similar Cy5-labeling efficiency on the same substrates.…”

“…In addition, we observed a higher FRET efficiency with the NS3h Y618AzF-Cy5 construct compared to a previously reported cysteine-labeled NS3h Cys-Cy5 construct, where site-specific labeling of a single cysteine could not be guaranteed. 28 The higher FRET with the NS3h Y618AzF-Cy5 construct, where we could ensure site-specific labeling, supports that Cy5 was attached at a better position/orientation on NS3h for FRET with the Cy3 dye located on the DNA substrate. 44 At the single-molecule level, we could detect FRET efficiencies at approximately 0.8 for binding events of NS3h Y618AzF-Cy5 to a Cy3-DNA substrate with a six-and sevenbase single-stranded loading region, regardless of the specific sequence.…”

“…HCV NS3h was expressed and purified as described previously. 28 Briefly, the coding sequence for HCV NS3h (genotype 1a) corresponding to amino acids 167−631 of NS3 was expressed in Escherichia coli BL21(DE3) cells with a C-terminal hexa-histidine tag. For incorporation of the unnatural amino acid, AzF was added at 1 mM in the bacterial culture medium and an orthogonal tRNA/ tRNA synthetase pair from the lab of Dr. Peter Schultz 29 was used to incorporate AzF in response to an amber stop codon introduced in the coding sequence of NS3h by site-directed mutagenesis.…”

“…Ensemble FRET measurements were conducted as described previously. 28 Briefly, 200 nM of the indicated Cy3-DNA substrate was incubated with 400 nM NS3h (unless otherwise stated) in NS3h binding buffer (25 mM MOPS, pH 6.0, 10 mM NaCl, 3 mM MgCl 2 , and 0.1% Tween 20). Experiments were conducted in 96-well roundbottom black plates (Greiner), and the ratiometric intensitybased FRET proximity ratio (E pr ) was determined upon irradiation at 520 nm using a SpectraMax i3 plate reader (Molecular Devices).…”

Conformational Changes Spanning Angstroms to Nanometers via a Combined Protein-Induced Fluorescence Enhancement–Förster Resonance Energy Transfer Method

“…To reconcile the two techniques, a quantitative method capable of simultaneously and independently measuring PIFE and FRET changes is required. In this regard, a number of studies have utilized PIFE and FRET methods in a parallel set of experiments in ensemble − and/or at the single-molecule level. − Ongoing work from our group on the dynamics of the hepatitis C virus nonstructural protein 5B (HCV-NS5B) and helicase (NS3) and that of others provided for motivation into decoupling contributions from PIFE and FRET such that ultimately both techniques may be utilized simultaneously. That is, through a single set of experiments, one may obtain information on both short-range dynamics, <3 nm, through PIFE and long-range dynamics, between 3 and 8 nm, through FRET.…”