title: Ligand-induced changes in hepatitis C virus NS5B polymerase structure.
abstract: Hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B) is required for viral replication. Crystal structures of the NS5B apoprotein show that the finger and thumb domains interact to encircle the active site, and that inhibitors defined by P495 resistance that bind to the thumb-finger interface displace the Δ1 finger loop and disrupt this structure. Since crystal structures may not reveal all of the conformations of a protein in solution we have developed an alternative method, using limited trypsin protease digestion, to investigate the impact of inhibitors as well as substrates on the movement of the Δ1 loop. This assay can be used to study NS5B under conditions that support enzymatic activity. In the absence of inhibitors, no specific region of NS5B was hypersensitive to trypsin, and no specific intermediate cleavage products were formed. Binding of P495-site inhibitors to NS5B induced specific trypsin hypersensitivity at lysine residues 50 and 51. Previously characterized inhibitors and mutant polymerases were used to link this specific trypsin hypersensitivity to movement of the Δ1 loop. Trypsin hypersensitivity identical to the inhibitor pattern was also induced by the binding of the RNA template. The addition of primer to the NS5B-template complex eliminated the hypersensitivity. The data are consistent with displacement of the Δ1 finger loop from the thumb by the binding of template, and reversal by the addition of primer or NTP. Our results complement inhibitor-enzyme co-crystal studies, and the assay provides a rapid and sensitive method to study dynamic changes in HCV NS5B polymerase conformation under conditions that support functional activity.
authors.affiliation: Department of Virology, Bristol-Myers Squibb Co., Research & Development, Wallingford, CT 06492, USA. karen.rigat@bms.com
authors.forename: Karen
authors.initials: K
authors.lastname: Rigat
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Hudyma
authors.forename: Min
authors.initials: M
authors.lastname: Ding
authors.forename: Xiaofan
authors.initials: X
authors.lastname: Zheng
authors.forename: Robert G
authors.initials: RG
authors.lastname: Gentles
authors.forename: Brett R
authors.initials: BR
authors.lastname: Beno
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.forename: Susan B
authors.initials: SB
authors.lastname: Roberts
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D008024:Ligands; D050505:Mutant Proteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D014357:Trypsin