Advertisement

Mechanisms of HCV NS3 Helicase Monitored by Optical Tweezers

  • Wei ChengEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

As one of the essential enzymes for viral genome replication, the hepatitis C virus NS3 helicase is one of the best characterized RNA helicases to date in understanding the mechanistic cycles in a helicase-catalyzed strand separation reaction. Recently, single-molecule studies on NS3, in particular the use of optical tweezers with sub-base pair spatial resolution, have allowed people to examine the potential elementary steps of NS3 in unwinding the double-stranded RNA fueled by ATP binding and hydrolysis. In this chapter, I detail the essential technical elements involved in conducting a high-resolution optical tweezers study of NS3 helicase, starting from the purification of the recombinant helicase protein from E. coli to setting up a high-resolution single-molecule experiment using optical tweezers.

Key words

RNA helicase Optical tweezers Hepatitis C virus NS3 Single-molecule 

Notes

Acknowledgments

This work was supported by NSF CAREER Award CHE1149670 to WC, NIH Director’s New Innovator Award 1DP2OD008693 to WC, and also in part by Research Grant No. 5-FY10-490 to WC from the March of Dimes Foundation.

References

  1. 1.
    Lohman TM, Bjornson KP (1996) Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem 65:169–214PubMedCrossRefGoogle Scholar
  2. 2.
    Pyle AM (2008) Translocation and unwinding mechanisms of RNA and DNA helicases. Annu Rev Biophys 37:317–336PubMedCrossRefGoogle Scholar
  3. 3.
    Rabhi M, Tuma R, Boudvillain M (2010) RNA remodeling by hexameric RNA helicases. RNA Biol 7:655–666PubMedCrossRefGoogle Scholar
  4. 4.
    Kolykhalov AA, Mihalik K, Feinstone SM, Rice CM (2000) Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo. J Virol 74:2046–2051PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Frick DN (2003) Helicases as antiviral drug targets. Drug News Perspect 16:355–362PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Raney KD, Sharma SD, Moustafa IM, Cameron CE (2010) Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target. J Biol Chem 22725–22731Google Scholar
  7. 7.
    Murray CL, Jones CT, Rice CM (2008) Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis. Nat Rev Microbiol 6:699–708PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Lindenbach BD, Rice CM (2005) Unravelling hepatitis C virus replication from genome to function. Nature 436:933–938PubMedCrossRefGoogle Scholar
  9. 9.
    Ma Y, Yates J, Liang Y et al (2008) NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol 82:7624–7639PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Gorbalenya AE, Koonin EV (1993) Helicases: amino acid sequence comparisons and structure-function relationships. Curr Opin Struct Biol 3:419–429CrossRefGoogle Scholar
  11. 11.
    Korolev S, Yao N, Lohman TM et al (1998) Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases. Protein Sci 7:605–610PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Pang PS, Jankowsky E, Planet PJ, Pyle AM (2002) The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J 21:1168–1176PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Serebrov V, Pyle AM (2004) Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430:476–480PubMedCrossRefGoogle Scholar
  14. 14.
    Cheng W, Dumont S, Tinoco I Jr, Bustamante C (2007) NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork. Proc Natl Acad Sci U S A 104:13954–13959PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Serebrov V, Beran RK, Pyle AM (2009) Establishing a mechanistic basis for the large kinetic steps of the NS3 helicase. J Biol Chem 284:2512–2521PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Jennings TA, Mackintosh SG, Harrison MK et al (2009) NS3 helicase from the hepatitis C virus can function as a monomer or oligomer depending on enzyme and substrate concentrations. J Biol Chem 284:4806–4814PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Cheng W, Arunajadai SG, Moffitt JR et al (2011) Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase. Science 333:1746–1749PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Myong S, Bruno MM, Pyle AM, Ha T (2007) Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317:513–516PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Dumont S, Cheng W, Serebrov V et al (2006) RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439:105–108PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Cheng W, Hou X, Ye F (2010) Use of tapered amplifier diode laser for biological-friendly high-resolution optical trapping. Opt Lett 35:2988–2990PubMedCrossRefGoogle Scholar
  21. 21.
    Bustamante C, Chemla YR, Moffitt JR (2009) High-resolution dual-trap optical tweezers with differential detection: instrument design. CSH Protoc, pdb ip73Google Scholar
  22. 22.
    Beran RK, Bruno MM, Bowers HA et al (2006) Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track. J Mol Biol 358:974–982PubMedCrossRefGoogle Scholar
  23. 23.
    Cheng W, Hsieh J, Brendza KM, Lohman TM (2001) E. coli Rep oligomers are required to initiate DNA unwinding in vitro. J Mol Biol 310:327–350PubMedCrossRefGoogle Scholar
  24. 24.
    Bjornson KP, Amaratunga M, Moore KJ, Lohman TM (1994) Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer. Biochemistry 33:14306–14316PubMedCrossRefGoogle Scholar
  25. 25.
    Arunajadai SG, Cheng W (2013) Step detection in single-molecule real time trajectories embedded in correlated noise. PLoS One 8:e59279PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Pharmaceutical SciencesUniversity of MichiganAnn ArborUSA
  2. 2.Department of BiophysicsUniversity of MichiganAnn ArborUSA

Personalised recommendations