title: Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase.
abstract: The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305.
authors.forename: Q May
authors.initials: QM
authors.lastname: Wang
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hockman
authors.forename: Kirk
authors.initials: K
authors.lastname: Staschke
authors.forename: Robert B
authors.initials: RB
authors.lastname: Johnson
authors.forename: Katharine A
authors.initials: KA
authors.lastname: Case
authors.forename: Jirong
authors.initials: J
authors.lastname: Lu
authors.forename: Steve
authors.initials: S
authors.lastname: Parsons
authors.forename: Faming
authors.initials: F
authors.lastname: Zhang
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Rathnachalam
authors.forename: Karla
authors.initials: K
authors.lastname: Kirkegaard
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Colacino
mesh_terms: D003432:Cross-Linking Reagents; D003460:Crystallization; D004795:Enzyme Stability; D016174:Hepacivirus / Q000201:enzymology*; D006358:Hot Temperature; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase