Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli.
The hepatitis C virus (HCV) represents a major public health problem that can produce liver failure and hepatocellular carcinoma in chronically infected patients. Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and initiate studies of its role in HCV genomic replication. In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activity. This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a template. The polymerase activity of recombinant NS5B was primer-dependent and was active for at least 60 min of incubation at 30 degrees C. Deletion of the carboxyl terminal region of HCV NS5B resulted in a loss of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme. Recombinant NS5B (RDRP) should assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors.
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Department of Medicine, Genetics Program of the Winship Cancer Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
Xie
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Wang
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Hagedorn
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CH
D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D011072:Poly U / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D011072:Poly U; D005914:Globins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase