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Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Nature volume 435, pages 374–379 (2005)Cite this article

Abstract

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population1. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase2,3, as well as a pivotal regulator of replication2,4 and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth5,6. NS5A is a large phosphoprotein (56–58 kDa) with an amphipathic α-helix at its amino terminus that promotes membrane association7,8,9. After this helix region, NS5A is organized into three domains10. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule10. Mutations disrupting either the membrane anchor7,8 or zinc binding10 of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-Å resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.

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Figure 1: An overview of the NS5A domain I structure.
Figure 2: The NS5A zinc-binding motif and disulphide bond.
Figure 3: Molecular surfaces of domain I.
Figure 4: The NS5A domain I dimer reveals potential interaction surfaces.

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References

  1. World Health Organization. Hepatitis C: global prevalence. Wkly. Epidemiol. Rec. 72, 341–344 (1997)

    Google Scholar 

  2. Blight, K. J., Kolykhalov, A. A. & Rice, C. M. Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974 (2000)

    Article  ADS  CAS  Google Scholar 

  3. Lohmann, V. et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113 (1999)

    Article  CAS  Google Scholar 

  4. Lohmann, V., Korner, F., Dobierzewska, A. & Bartenschlager, R. Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J. Virol. 75, 1437–1449 (2001)

    Article  CAS  Google Scholar 

  5. Tellinghuisen, T. L. & Rice, C. M. Interaction between hepatitis C virus proteins and host cell factors. Curr. Opin. Microbiol. 5, 419–427 (2002)

    Article  CAS  Google Scholar 

  6. Macdonald, A. & Harris, M. Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen. Virol. 85, 2485–2502 (2004)

    Article  CAS  Google Scholar 

  7. Elazar, M. et al. Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication. J. Virol. 77, 6055–6061 (2003)

    Article  CAS  Google Scholar 

  8. Penin, F. et al. Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 279, 40835–40843 (2004)

    Article  CAS  Google Scholar 

  9. Brass, V. et al. An amino-terminal amphipathic α-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 277, 8130–8139 (2002)

    Article  CAS  Google Scholar 

  10. Tellinghuisen, T. L., Marcotrigiano, J., Gorbalenya, A. E. & Rice, C. M. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J. Biol. Chem. 279, 48576–48587 (2004)

    Article  CAS  Google Scholar 

  11. Holm, L. & Sander, C. Mapping the protein universe. Science 273, 595–603 (1996)

    Article  ADS  CAS  Google Scholar 

  12. Alberts, I. L., Nadassy, K. & Wodak, S. J. Analysis of zinc binding sites in protein crystal structures. Protein Sci. 7, 1700–1716 (1998)

    Article  CAS  Google Scholar 

  13. Engh, R. & Huber, R. Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallogr. A 47, 392–400 (1991)

    Article  Google Scholar 

  14. Kuiken, C., Yusim, K., Boykin, L. & Richardson, R. The Los Alamos HCV sequence database. Bioinformatics 21, 379–384 (2005)

    Article  CAS  Google Scholar 

  15. Jones, S. & Thornton, J. M. Principles of protein–protein interactions. Proc. Natl Acad. Sci. USA 93, 13–20 (1996)

    Article  ADS  CAS  Google Scholar 

  16. Dimitrova, M., Imbert, I., Kieny, M. P. & Schuster, C. Protein–protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 77, 5401–5414 (2003)

    Article  CAS  Google Scholar 

  17. Wang, Q. M. et al. Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase. J. Virol. 76, 3865–3872 (2002)

    Article  CAS  Google Scholar 

  18. Huang, L. et al. Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli. Protein Expr. Purif. 37, 144–153 (2004)

    Article  CAS  Google Scholar 

  19. Otwinowski, Z. & Minor, M. in Methods in Enzymology Vol. 276, Macromolecular Crystallography, Part A (eds Carter, C. W. & Sweet, R. M.) 307–326 (Academic, New York, 1997)

    Book  Google Scholar 

  20. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999)

    Article  CAS  Google Scholar 

  21. Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994)

    Article  Google Scholar 

  22. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  23. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  24. Laskowski, R. A., Moss, D. S. & Thornton, J. M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993)

    Article  CAS  Google Scholar 

  25. Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983)

    Article  CAS  Google Scholar 

  26. DeLano, W. L. The PyMOL molecular graphics system. http://www.pymol.org (2002).

  27. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    Article  ADS  CAS  Google Scholar 

  28. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882 (1997)

    Article  Google Scholar 

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Acknowledgements

We would like to acknowledge R. MacKinnon, S. Darst and H. Mueller for the use of X-ray diffractometers, related equipment and software. We appreciate access to beamline X9A at the National Synchrotron Light Source (NSLS) at the Brookhaven National Labs and acknowledge the assistance of NSLS staff. J.-W. Carroll provided vital assistance with data collection. D. Jeruzalmi provided the program msf_similarity_to_pdb. We wish to thank S. Darst, M. Evans, A. Gauthier, C. Jones, B. Lindenbach, I. Lorenz, T. von Hahn, M. Tellinghuisen and K. Tellinghuisen for critical reading of this manuscript. T.L.T. was supported in part by fellowships from the Charles H. Revson Foundation for Biomedical Research and the National Institutes of Health Ruth L. Kirschstein National Research Service Award, granted through the National Institute of Allergy and Infectious Disease. J.M. was supported as a Merck Fellow of the Life Sciences Research Foundation. Additional financial support for this work came from grants from the National Institutes of Health and the Greenberg Medical Research Institute (C.M.R.).Author Contributions T.L.T., J.M. and C.M.R. conceived these experiments. T.L.T. generated all reagents, materials, proteins and crystals used herein, with assistance from J.M. All data collection was carried out by J.M. and T.L.T. Data processing, model building and refinement were performed by T.L.T., with significant input and assistance from J.M. The manuscript was written by T.L.T. with comments and assistance from J.M. and C.M.R.

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Authors and Affiliations

  1. Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Avenue, Box 64, New York, New York, 10021, USA

    Timothy L. Tellinghuisen, Joseph Marcotrigiano & Charles M. Rice

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  1. Timothy L. Tellinghuisen
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  3. Charles M. Rice
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Correspondence to Joseph Marcotrigiano or Charles M. Rice.

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Competing interests

The coordinates for this structure have been deposited in the Protein Data Bank (PDB) under accession code 1ZH1. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Sequence map of NS5A domain I. Secondary structures are shown and numbered as in the structure. Amino acids are coloured as follows: red, cysteines contacting zinc; green, disulphide bonded cysteines; grey, proline subdomain connector; blue, residues lining the basic groove; pink, dimer interface residues; and orange, conserved surface patch. (JPG 54 kb)

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Tellinghuisen, T., Marcotrigiano, J. & Rice, C. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435, 374–379 (2005). https://doi.org/10.1038/nature03580

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