title: Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation.
abstract: Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information.
authors: From the Department of Molecular Biosciences, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712.
authors: Jiawen
authors: J
authors: Li
authors: From the Department of Molecular Biosciences, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712 kajohnson@mail.utexas.edu.
authors: Kenneth A
authors: KA
authors: Johnson
mesh_terms: D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology*; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D004791:Enzyme Inhibitors; D009711:Nucleotides; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: NS5B; RNA initiation; computer modeling; enzyme kinetics; hepatitis C virus (HCV); inhibition mechanism; non nucleoside inhibitors; viral polymerase; viral replication