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J Biol Chem, Vol. 273, Issue 25, 15479-15486, June 19, 1998
From the The hepatitis C virus (HCV) NS5B protein
encodes an RNA-dependent RNA polymerase (RdRP), which is
the central catalytic enzyme of HCV replicase. We established a new
method to purify soluble HCV NS5B in the glutathione
S-transferase-fused form NS5Bt from Escherichia
coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The
recombinant soluble protein exhibited RdRP activity in
vitro which was dependent upon the template and primer, but it
did not exhibit the terminal transferase activity that has been
reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione
S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt
shares most of the properties. Substitution mutations of NS5Bt at the
GDD motif, which is highly conserved among viral RdRPs, and at the
clustered basic residues (amino acids 2919-2924 and 2693-2699)
abolished the RdRP activity. The C-terminal region of NS5B, which is
dispensable for the RdRP activity, dramatically affected the
subcellular localization of NS5B retaining it in perinuclear sites in
transiently overexpressed mammalian cells. These results may provide
some clues to dissecting the molecular mechanism of the HCV replication
and also act as a basis for developing new anti-viral drugs.
RNA-dependent RNA Polymerase Activity of the Soluble
Recombinant Hepatitis C Virus NS5B Protein Truncated at the
C-terminal Region
§,§,,,
Department of Molecular Oncology,
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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