title: Molecular and structural basis for the roles of hepatitis C virus polymerase NS5B amino acids 15, 223, and 321 in viral replication and drug resistance.
abstract: Resistance to the 2'-F-2'-C-methylguanosine monophosphate nucleotide hepatitis C virus (HCV) inhibitors PSI-352938 and PSI-353661 was associated with a combination of amino acid changes (changes of S to G at position 15 [S15G], C223H, and V321I) within the genotype 2a nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase. To understand the role of these residues in viral replication, we examined the effects of single and multiple point mutations on replication fitness and inhibition by a series of nucleotide analog inhibitors. An acidic residue at position 15 reduced replicon fitness, consistent with its proximity to the RNA template. A change of the residue at position 223 to an acidic or large residue reduced replicon fitness, consistent with its proposed proximity to the incoming nucleoside triphosphate (NTP). A change of the residue at position 321 to a charged residue was not tolerated, consistent with its position within a hydrophobic cavity. This triple resistance mutation was specific to both genotype 2a virus and 2'-F-2'-C-methylguanosine inhibitors. A crystal structure of the NS5B S15G/C223H/V321I mutant of the JFH-1 isolate exhibited rearrangement to a conformation potentially consistent with short primer-template RNA binding, which could suggest a mechanism of resistance accomplished through a change in the NS5B conformation, which was better tolerated by genotype 2a virus than by viruses of other genotypes.
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA alam@noviratherapeutics.com tedwards@be4.com.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Lam
authors.affiliation: Beryllium, Bainbridge Island, Washington, USA alam@noviratherapeutics.com tedwards@be4.com.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Edwards
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA.
authors.forename: Ralph T
authors.initials: RT
authors.lastname: Mosley
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA Gilead Sciences, Foster City, California, USA.
authors.forename: Eisuke
authors.initials: E
authors.lastname: Murakami
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA.
authors.forename: Shalini
authors.initials: S
authors.lastname: Bansal
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Lugo
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA.
authors.forename: Haiying
authors.initials: H
authors.lastname: Bao
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Otto
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sofia
authors.affiliation: Pharmasset, Inc., Princeton, New Jersey, USA.
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Furman
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003498:Cyclic P-Oxides / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000235:genetics*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D009705:Nucleosides / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000648:ultrastructure*; D014779:Virus Replication / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D003498:Cyclic P-Oxides; D006157:Guanosine Monophosphate; D009705:Nucleosides; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C562230:2-((5-(2-amino-6-methoxypurin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy)phenoxyphosphorylamino)propionic acid isopropyl ester; C556498:PSI 352938; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase