Intracellular hepatitis c virus RNA-dependent RNA polymerase activity

Studies of intracellular hepatitis C virus (HCV) RNA-dependent RNA polymera se activity (RdRp activity) have been limited by the poor replicative capac ity 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 polymerise and its template s under the control of the T7 promoter in the presence of an infection with recombinant vaccinia virus (vTF7-3) expressing the bacteriophage T7 DNA-de pendent RNA polymerase. Both negative-strand and positive-strand RNA synthe sis were characterised, and the role of the other HCV non-structural protei ns for polymerase activity was assessed. With this assay we were able to sh ow that: a) Intracellular HCV RdRp activity is not restricted to, but is hi gher for templates containing HCV specific sequences, b) The HCV polymerise is active within the polyprotein precursor, c) Cleavage of NS5b from the p olyprotein precursor does not determine template specificity, and d) HCV Rd Rp activity is higher in the presence of the other HCV non-structural prote ins and lower within a protease-deficient polyprotein precursor. This metho d allows the measurement of intracellular HCV polymerase activity and may b e used to test substances against the HCV polymerise in search of potential drugs for anti-HCV therapy.