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

Area of Science:

• Biochemistry
• Virology
• Molecular Biology

Background:

• Hepatitis C virus nonstructural protein 3 (NS3) C-terminal helicase is crucial for viral replication.
• Understanding its interaction with nucleic acid substrates is key to inhibiting viral activity.

Purpose of the Study:

• To investigate the dynamics of NS3 helicase binding and movement on nucleic acid substrates.
• To elucidate the mechanism preceding the DNA unwinding process.

Main Methods:

• Utilized ensemble Förster resonance energy transfer (FRET) and protein induced fluorescence enhancement (PIFE) assays.
• Employed single-molecule PIFE experiments to analyze enzyme-nucleic acid interactions at high resolution.

Main Results:

• NS3 helicase binds to the 3' single-stranded overhang of DNA substrates.
• Single-molecule PIFE revealed three distinct binding sites and enzyme translocation between them.
• A W501A mutant, lacking DNA stacking ability, exhibited significantly reduced switching between binding sites.

Conclusions:

• The NS3 helicase exhibits ATP-independent random binding and sliding along the DNA overhang.
• The enzyme can move between adjacent nucleotide binding sites without dissociation.
• Stacking interactions mediated by W501 are critical for NS3 helicase dynamics and translocation.