Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase

Structure. 1999 Nov 15;7(11):1353-63. doi: 10.1016/s0969-2126(00)80025-8.


Background: Hepatitis C virus (HCV) currently infects approximately 3% of the world's population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown.

Results: We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site.

Conclusions: The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.

MeSH terms

  • Amino Acid Sequence
  • Crystallography, X-Ray
  • Hepacivirus / enzymology*
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Processing, Post-Translational*
  • RNA Helicases / chemistry
  • RNA Helicases / metabolism*
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / metabolism*
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / metabolism*


  • NS3 protein, hepatitis C virus
  • Viral Nonstructural Proteins
  • Serine Endopeptidases
  • RNA Helicases

Associated data

  • PDB/1CU1