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PMID: 9882374 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli.

Journal of virology ·Vol. 73 ·No. 2 ·1999-02-00 ·Pages 1649-54

Ferrari E, Wright-Minogue J, Fang JW, Baroudy BM, Lau JY, Hong Z

Abstract

Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

MeSH Terms
Amino Acid Sequence Cations, Divalent Enzyme Inhibitors/pharmacology Escherichia coli Gliotoxin/pharmacology Hepacivirus/enzymology Humans Metals Molecular Sequence Data RNA-Dependent RNA Polymerase/antagonists & inhibitors,genetics,isolation & purification,metabolism Recombinant Fusion Proteins/genetics,isolation & purification,metabolism Sequence Deletion Solubility Viral Nonstructural Proteins/antagonists & inhibitors,genetics,isolation & purification,metabolism
Chemicals
Cations, Divalent Enzyme Inhibitors Metals NS-5 protein, hepatitis C virus Recombinant Fusion Proteins Viral Nonstructural Proteins Gliotoxin RNA-Dependent RNA Polymerase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Ferrari E
Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.
Wright-Minogue J
Fang J W
Baroudy B M
Lau J Y
Hong Z
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.73.2.1649-1654.1999
Published
1999-02-00
Pages
1649-54
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC103993
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.73.2.1649-1654.1999 PMC Full Text View journal details
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