Carola Berger1, Inés Romero-Brey1, Danijela Radujkovic1, Raphael Terreux2, Margarita Zayas1, David Paul1, Christian Harak1, Simone Hoppe1, Min Gao3, Francois Penin4, Volker Lohmann1, Ralf Bartenschlager5. 1. Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany. 2. CNRS, UMR5086, Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, Lyon, France; Labex Ecofect (ANR-11-LABX-0042), University of Lyon, Lyon, France; Faculté de Pharmacie (ISPB), Lyon, France. 3. Bristol-Myers Squibb Research and Development, Wallingford, Connecticut. 4. CNRS, UMR5086, Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, Lyon, France; Labex Ecofect (ANR-11-LABX-0042), University of Lyon, Lyon, France. 5. Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Centre for Infection Research, Heidelberg University, Heidelberg, Germany. Electronic address: Ralf_Bartenschlager@med.uni-heidelberg.de.
Abstract
BACKGROUND & AIMS: Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. METHODS: We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. RESULTS: NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. CONCLUSIONS: Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy.
BACKGROUND & AIMS: Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. METHODS: We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. RESULTS: NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. CONCLUSIONS: Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy.
Authors: David R McGivern; Takahiro Masaki; William Lovell; Chris Hamlett; Susanne Saalau-Bethell; Brent Graham Journal: J Virol Date: 2015-03-04 Impact factor: 5.103
Authors: Christian Schenk; Max Meyrath; Uwe Warnken; Martina Schnölzer; Walter Mier; Christian Harak; Volker Lohmann Journal: J Virol Date: 2018-11-27 Impact factor: 5.103