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Hepatitis C Virus NS5B RNA Replicase Specifically Binds Ribosomes
Author(s) -
Tanaka Torahiko,
Sugiyama Kazuo,
Ikeda Masanori,
Naganuma Atsushi,
Nozaki Akito,
Saito Masaki,
Shimotohno Kunitada,
Kato Nobuyuki
Publication year - 2000
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2000.tb02532.x
Subject(s) - ns5b , ribosome , biology , rna dependent rna polymerase , biochemistry , rna , amino acid , bacteriophage ms2 , microbiology and biotechnology , virology , virus , hepatitis c virus , hepacivirus , gene , coat protein
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 2 μ m 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.