The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNA-dependent RNA polymerase.
We have previously determined the crystal structure of a non-structural 5B (NS5B) protein, an RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). NS5B protein with the hydrophobic C-terminal 21 amino acid residues truncated, designated NS5B(570), shows a typical nucleotide polymerase structure resembling a right-hand shape. In the crystal structure, a C-terminal region between Leu545 and His562 occupies a putative RNA-binding cleft of this polymerase and seems to inhibit the polymerase activity. Varieties of recombinant NS5B proteins (NS5B(552), NS5B(544), NS5B(536) or NS5B(531), with C-terminal 39, 47, 55 or 60 amino acid residues truncated, respectively) were systematically constructed to elucidate effects of the region on the polymerase activity. NS5B(544), NS5B(536) and NS5B(531) showed markedly higher RdRp activities compared to the activities of NS5B(570) or NS5B(552). Furthermore, when the hydrophobic amino acid residues Leu547, Trp550 and Phe551 (LWF) in NS5B(570) and NS5B(552) were changed to alanine, their activities were higher than that of the original NS5B(570). The crystal structures of the various recombinant NS5B proteins were also determined. Structural comparison of the NS5B proteins indicates that the activation was caused by elimination of a unique hydrophobic interaction between the three C-terminal residues and a shallowly concave pocket consisting of thumb and palm domains.
Adachi
Tsuyoshi
T
Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan. tsuyoshi.adachi@ims.jti.co.jp
Ago
Hideo
H
Habuka
Noriyuki
N
Okuda
Kayo
K
Komatsu
Masakazu
M
Ikeda
Satoru
S
Yatsunami
Kimio
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase