Intracellular hepatitis C virus RNA-dependent RNA polymerase activity.
Studies of intracellular hepatitis C virus (HCV) RNA-dependent RNA polymerase activity (RdRp activity) have been limited by the poor replicative capacity of HCV in cell culture. We have developed a method that allows for the measurement of HCV specific RdRp activity in eukaryotic cells. This method is based on the transient expression of the HCV polymerase and its templates under the control of the T7 promoter in the presence of an infection with recombinant vaccinia virus (vTF7-3) expressing the bacteriophage T7 DNA-dependent RNA polymerase. Both negative-strand and positive-strand RNA synthesis were characterised, and the role of the other HCV non-structural proteins for polymerase activity was assessed. With this assay we were able to show that: a) Intracellular HCV RdRp activity is not restricted to, but is higher for templates containing HCV specific sequences, b) The HCV polymerase is active within the polyprotein precursor, c) Cleavage of NS5b from the polyprotein precursor does not determine template specificity, and d) HCV RdRp activity is higher in the presence of the other HCV non-structural proteins and lower within a protease-deficient polyprotein precursor. This method allows the measurement of intracellular HCV polymerase activity and may be used to test substances against the HCV polymerase in search of potential drugs for anti-HCV therapy.
Goobar-Larsson
L
L
Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Sweden.
Wiklund
L
L
Schwartz
S
S
D006367:HeLa Cells; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism
D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase