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

13 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
Publication statusPublished - 2000

Fingerprint

Viral Structural Proteins
RNA Replicase
Ribosomes
Hepacivirus
Proteins
Amylose
Amino Acids
23S Ribosomal RNA
Poly A-U
16S Ribosomal RNA
Maltose-Binding Proteins
Uridine Monophosphate
Uridine Triphosphate
Viral Genome
Ultracentrifugation
Life Cycle Stages
Centrifugation
Escherichia coli

Keywords

  • Hepatitis C virus
  • Ribosomes
  • RNA replicase

ASJC Scopus subject areas

  • Immunology and Microbiology(all)
  • Microbiology (medical)
  • Microbiology

Cite this

Tanaka, T., Sugiyama, K., Ikeda, M., Naganuma, A., Nozaki, A., Saito, M., ... Kato, N. (2000). Hepatitis C virus NS5B RNA replicase specifically binds ribosomes. Microbiology and Immunology, 44(6), 543-550.

Hepatitis C virus NS5B RNA replicase specifically binds ribosomes. / Tanaka, Torahiko; Sugiyama, Kazuo; Ikeda, Masanori; Naganuma, Atsushi; Nozaki, Akito; Saito, Masaki; Shimotohno, Kunitada; Kato, Nobuyuki.

In: Microbiology and Immunology, Vol. 44, No. 6, 2000, p. 543-550.

Research output: Contribution to journalArticle

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, vol. 44, no. 6, pp. 543-550.
Tanaka T, Sugiyama K, Ikeda M, Naganuma A, Nozaki A, Saito M et al. Hepatitis C virus NS5B RNA replicase specifically binds ribosomes. Microbiology and Immunology. 2000;44(6):543-550.
Tanaka, Torahiko ; Sugiyama, Kazuo ; Ikeda, Masanori ; Naganuma, Atsushi ; Nozaki, Akito ; Saito, Masaki ; Shimotohno, Kunitada ; Kato, Nobuyuki. / Hepatitis C virus NS5B RNA replicase specifically binds ribosomes. In: Microbiology and Immunology. 2000 ; Vol. 44, No. 6. pp. 543-550.
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