RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region.
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.
Yamashita
T
T
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-Machi, Kanazawa, Ishikawa, Japan.
Kaneko
S
S
Shirota
Y
Y
Qin
W
W
Nomura
T
T
Kobayashi
K
K
Murakami
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D005982:Glutathione Transferase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D013917:Thrombin