title: A single-amino-acid mutation in hepatitis C virus NS5A disrupting FKBP8 interaction impairs viral replication.
abstract: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) regulates viral replication through its interaction with host and other viral proteins. We have previously shown that FK506-binding protein 8 (FKBP8) binds to NS5A and recruits Hsp90 to form a complex that participates in the replication of HCV. In this study, we examined the biochemical characteristics of the interaction and the intracellular localization of NS5A and FKBP8. Surface plasmon resonance analysis revealed that the dissociation constant of the interaction between the purified FKBP8 and NS5A expressed in bacteria was 82 nM. Mutational analyses of NS5A revealed that a single amino acid residue of Val or Ile at position 121, which is well conserved among all genotypes of HCV, is critical for the specific interaction with FKBP8. Substitution of the Val(121) to Ala drastically impaired the replication of HCV replicon cells, and the drug-resistant replicon cells emerging after drug selection were shown to have reverted to the original arrangement by replacing Ala(121) with Val. Examination of individual fields of the replicon cells by both fluorescence microscopy and electron microscopy (the correlative fluorescence microscopy-electron microscopy technique) revealed that FKBP8 is partially colocalized with NS5A in the cytoplasmic structure known as the membranous web. These results suggest that specific interaction of NS5A with FKBP8 in the cytoplasmic compartment plays a crucial role in the replication of HCV.
authors.affiliation: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita-shi, Osaka 565-0871, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Okamoto
authors.forename: Hiroko
authors.initials: H
authors.lastname: Omori
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Kaname
authors.forename: Takayuki
authors.initials: T
authors.lastname: Abe
authors.forename: Yorihiro
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Miyamura
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Yoshimori
authors.forename: Kohji
authors.initials: K
authors.lastname: Moriishi
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Matsuura
mesh_terms: D019943:Amino Acid Substitution; D003593:Cytoplasm / Q000737:chemistry; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D020125:Mutation, Missense; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D022021:Tacrolimus Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D011994:Recombinant Proteins; D022021:Tacrolimus Binding Proteins; D017361:Viral Nonstructural Proteins; C471205:FKBP8 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase