Hepatitis C virus NS5B RNA replicase specifically binds ribosomes

Torahiko Tanaka, Kazuo Sugiyama, Masanori Ikeda, Atsushi Naganuma, Akito Nozaki, Masaki Saito, Kunitada Shimotohno, Nobuyuki Kato

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 μm 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.

Original languageEnglish
Pages (from-to)543-550
Number of pages8
JournalMICROBIOLOGY and IMMUNOLOGY
Volume44
Issue number6
DOIs
Publication statusPublished - Jan 1 2000

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Keywords

  • Hepatitis C virus
  • RNA replicase
  • Ribosomes

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Virology

Cite this

Tanaka, T., Sugiyama, K., Ikeda, M., Naganuma, A., Nozaki, A., Saito, M., Shimotohno, K., & Kato, N. (2000). Hepatitis C virus NS5B RNA replicase specifically binds ribosomes. MICROBIOLOGY and IMMUNOLOGY, 44(6), 543-550. https://doi.org/10.1111/j.1348-0421.2000.tb02532.x