title: NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase IIIα complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes.
abstract: The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex.
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Reghellin
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Donnici
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Fenu
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Berno
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano Bicocca, Milan, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Calabrese
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Pagani
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Abrignani
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano Bicocca, Milan, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Peri
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy defrancesco@ingm.org neddermann@ingm.org.
authors.forename: R
authors.initials: R
authors.lastname: De Francesco
authors.affiliation: Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy defrancesco@ingm.org neddermann@ingm.org.
authors.forename: P
authors.initials: P
authors.lastname: Neddermann
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure / Q000821:virology; D002784:Cholesterol / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D016174:Hepacivirus / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology; D022781:Hepatocytes / Q000187:drug effects / Q000648:ultrastructure / Q000821:virology; D006801:Humans; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002784:Cholesterol; D004791:Enzyme Inhibitors; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates; D017853:Phosphotransferases (Alcohol Group Acceptor); D017361:Viral Nonstructural Proteins; C037178:phosphatidylinositol 4-phosphate; C402182:phosphatidylinositol phosphate 4-kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase