Fuel Specificity of the Hepatitis C Virus NS3 Helicase

Craig A. Belon, David N. Frick

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3′-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson-Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2′ and 3′ carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn2+ supporting about 10 times faster unwinding than Mg2+. Unlike Mg2+, Mn2+ does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.

Original languageEnglish (US)
Pages (from-to)851-864
Number of pages14
JournalJournal of Molecular Biology
Issue number4
StatePublished - May 15 2009
Externally publishedYes


  • ATPase
  • DNA
  • motor protein
  • RNA
  • viral replication

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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