Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain.
Hepatitis C virus (HCV) JFH1 efficiently replicates and produces infectious virus particles in cultured cells. We compared polymerase activity between JFH1 and 1b strains in vitro. The RNA polymerase activity of 1b was 6.4% of that of JFH1. In order to study the mechanism and identify domains responsible for the high polymerase activity of JFH1, we converted the amino acids of 1b RdRp to those of JFH1, and compared their Km, Vmax and template binding activity. Four amino acid mutations in the thumb domain of 1b RdRp, S377R, A450S, E455N and Y561F increased 1b polymerase activity, and their activity was 23.1, 45.8, 28.9, and 36.1% of JFH1, respectively. Vmax and RNA binding activity of JFH1, 1bwt and 1bA450S was JFH1 > 1bA450S > 1b, which indicated both high processivity and slightly higher template binding activity contributed to the high polymerase activity of JFH1.
Weng
Leiyun
L
Unit of Viral Genome Regulation, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 225 South Chongqing Road, 200025 Shanghai, People's Republic of China.
Du
Jiamu
J
Zhou
Jingling
J
Ding
Jianping
J
Wakita
Takaji
T
Kohara
Michinori
M
Toyoda
Tetsuya
T