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

ACS Infect Dis. 2017 Jan 13;3(1):99-109. doi: 10.1021/acsinfecdis.6b00177. Epub 2016 Nov 23.

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.

Keywords: DNA helicase; RNA helicase; fluorescence resonance energy transfer (FRET); hepatitis C virus (HCV); protein induced fluorescence enhancement (PIFE).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Helicases / metabolism
  • Genotype
  • Hepacivirus / enzymology*
  • Hepacivirus / genetics
  • Nucleic Acids / chemistry
  • Nucleic Acids / metabolism*
  • Protein Binding
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*

Substances

  • NS3 protein, hepatitis C virus
  • Nucleic Acids
  • Viral Nonstructural Proteins
  • DNA Helicases

Grant support