title: A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin.
abstract: The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2'-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2'-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
authors.forename: Anupriya S
authors.initials: AS
authors.lastname: Kulkarni
authors.affiliation: Department of Chemistry, McGill University, Montreal, Quebec, Canada.
authors.forename: Masad J
authors.initials: MJ
authors.lastname: Damha
authors.affiliation: Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia, USA.
authors.forename: Raymond F
authors.initials: RF
authors.lastname: Schinazi
authors.affiliation: Gilead Sciences, Inc., Foster City, California, USA.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Mo
authors.affiliation: Gilead Sciences, Inc., Foster City, California, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Doehle
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
authors.forename: Selena M
authors.initials: SM
authors.lastname: Sagan
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada Department of Biochemistry, McGill University, Montreal, Quebec, Canada Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada gotte@ualberta.ca.
authors.forename: Matthias
authors.initials: M
authors.lastname: Götte
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016174:Hepacivirus / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D006860:Hydrogen Bonding; D008826:Microbial Sensitivity Tests; D009154:Mutation; D011485:Protein Binding; D012367:RNA, Viral / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012254:Ribavirin / Q000737:chemistry / Q000494:pharmacology*; D000069474:Sofosbuvir / Q000737:chemistry / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D011994:Recombinant Proteins; D012254:Ribavirin; D000069474:Sofosbuvir; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
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