title: Geneticin Stabilizes the Open Conformation of the 5' Region of Hepatitis C Virus RNA and Inhibits Viral Replication.
abstract: The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.
authors.affiliation: Instituto de Parasitología y Biomedicina López-Neyra CSIC, Granada, Spain CIBERehd Centro de Investigación Biomédica en RED de Enfermedades Hepáticas y Digestivas (ISCIII), Madrid, Spain.
authors.forename: Ascensión
authors.initials: A
authors.lastname: Ariza-Mateos
authors.affiliation: Instituto de Parasitología y Biomedicina López-Neyra CSIC, Granada, Spain CIBERehd Centro de Investigación Biomédica en RED de Enfermedades Hepáticas y Digestivas (ISCIII), Madrid, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Díaz-Toledano
authors.affiliation: Baruch S. Blumberg Institute, Doylestown, Pennsylvania, USA.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Block
authors.affiliation: Instituto de Parasitología y Biomedicina López-Neyra CSIC, Granada, Spain.
authors.forename: Samuel
authors.initials: S
authors.lastname: Prieto-Vega
authors.affiliation: Department of Pharmacology, Weill Medical College of Cornell University, New York, New York, USA jgomez@ipb.csic.es alisbirk@verizon.net.
authors.forename: Alex
authors.initials: A
authors.lastname: Birk
authors.affiliation: Instituto de Parasitología y Biomedicina López-Neyra CSIC, Granada, Spain CIBERehd Centro de Investigación Biomédica en RED de Enfermedades Hepáticas y Digestivas (ISCIII), Madrid, Spain jgomez@ipb.csic.es alisbirk@verizon.net.
authors.forename: Jordi
authors.initials: J
authors.lastname: Gómez
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor / Q000821:virology; D018387:Codon, Initiator; D005839:Gentamicins / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D018387:Codon, Initiator; D005839:Gentamicins; D012367:RNA, Viral; D043244:Ribonuclease III; D043262:Ribonuclease P; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C010680:antibiotic G 418