Hepatitis C Virus NS5B RNA Replicase Specifically Binds Ribosomes

J-STAGE
  • Tanaka Torahiko
    Virology and Glycobiology Division, National Cancer Center Researh Institute
  • Sugiyama Kazuo
    Virology and Glycobiology Division, National Cancer Center Researh Institute
  • Ikeda Masanori
    Virology and Glycobiology Division, National Cancer Center Researh Institute
  • Naganuma Atsushi
    Virology and Glycobiology Division, National Cancer Center Researh Institute
  • Nozaki Akito
    Virology and Glycobiology Division, National Cancer Center Researh Institute
  • Saito Masaki
    Virology and Glycobiology Division, National Cancer Center Researh Institute
  • Shimotohno Kunitada
    Department of Viral Oncology, Laboratory of Human Tumor Viruses, The Institute for Virus Research, Kyoto University
  • Kato Nobuyuki
    Virology and Glycobiology Division, National Cancer Center Researh Institute

抄録

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×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 2μM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160, 000×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.

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