title: Hepatitis C virus NS5B RNA replicase specifically binds ribosomes.
abstract: Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase. We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B). MBP-NS5B was recovered in the soluble fraction after centrifugation at 40,000 x g and affinity-purified with amylose resin. The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2 microM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160,000 x g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B. Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin. Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.
authors.affiliation: Virology and Glycobiology Division, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Sugiyama
authors.forename: M
authors.initials: M
authors.lastname: Ikeda
authors.forename: A
authors.initials: A
authors.lastname: Naganuma
authors.forename: A
authors.initials: A
authors.lastname: Nozaki
authors.forename: M
authors.initials: M
authors.lastname: Saito
authors.forename: K
authors.initials: K
authors.lastname: Shimotohno
authors.forename: N
authors.initials: N
authors.lastname: Kato
mesh_terms: D015870:Gene Expression; D006367:HeLa Cells; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase