title: The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization.
abstract: Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Dike
authors.initials: D
authors.lastname: Qiu
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Lemm
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Donald R
authors.initials: DR
authors.lastname: O'Boyle
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Jin-Hua
authors.initials: JH
authors.lastname: Sun
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Nower
authors.affiliation: Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Van
authors.initials: V
authors.lastname: Nguyen
authors.affiliation: Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Hamann
authors.affiliation: Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Snyder
authors.affiliation: Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Deon
authors.affiliation: Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Ruediger
authors.affiliation: Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Meanwell
authors.affiliation: Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Makonen
authors.initials: M
authors.lastname: Belema
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.affiliation: Department of Virology, Bristol-Myers Squibb Research and Development, Wallingford, CT, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fridell
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology*; D010766:Phosphorylation; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase