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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
World Health Organization. Hepatitis C: global prevalence. Wkly. Epidemiol. Rec. 72, 341–344 (1997)
Blight, K. J., Kolykhalov, A. A. & Rice, C. M. Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974 (2000)
Lohmann, V. et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113 (1999)
Lohmann, V., Korner, F., Dobierzewska, A. & Bartenschlager, R. Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J. Virol. 75, 1437–1449 (2001)
Tellinghuisen, T. L. & Rice, C. M. Interaction between hepatitis C virus proteins and host cell factors. Curr. Opin. Microbiol. 5, 419–427 (2002)
Macdonald, A. & Harris, M. Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen. Virol. 85, 2485–2502 (2004)
Elazar, M. et al. Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication. J. Virol. 77, 6055–6061 (2003)
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)
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)
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)
Holm, L. & Sander, C. Mapping the protein universe. Science 273, 595–603 (1996)
Alberts, I. L., Nadassy, K. & Wodak, S. J. Analysis of zinc binding sites in protein crystal structures. Protein Sci. 7, 1700–1716 (1998)
Engh, R. & Huber, R. Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallogr. A 47, 392–400 (1991)
Kuiken, C., Yusim, K., Boykin, L. & Richardson, R. The Los Alamos HCV sequence database. Bioinformatics 21, 379–384 (2005)
Jones, S. & Thornton, J. M. Principles of protein–protein interactions. Proc. Natl Acad. Sci. USA 93, 13–20 (1996)
Dimitrova, M., Imbert, I., Kieny, M. P. & Schuster, C. Protein–protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 77, 5401–5414 (2003)
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)
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)
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)
Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999)
Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994)
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)
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)
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)
Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983)
DeLano, W. L. The PyMOL molecular graphics system. http://www.pymol.org (2002).
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)
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)
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.
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.
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)
About this article
Cite this article
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
Trends in Pharmacological Sciences (2020)
Antiviral Research (2020)
QSAR studies on hepatitis C virus NS5A protein tetracyclic inhibitors in wild type and mutants by CoMFA and CoMSIA
SAR and QSAR in Environmental Research (2020)
Bioorganic & Medicinal Chemistry Letters (2020)