Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells

The influenza virus RNA polymerase (RdRp) was purified from insect cells (around 0.2 mg/l). The RdRp catalyzed all the biochemical reactions of influenza virus transcription and replication in vitro; dinucleotide ApG and globin mRNA-primed transcription, de novo initiation (replication), and polyadenylation. The optimal Mg concentration, pH and temperature were 8 mM, 8.0 and 25 C, respectively, which were slightly different from those measured for RdRp of virions. This system is a single-round transcription system. Km (lM) were 10.74 ± 0.26 (GTP), 33.22 ± 3.37 (ATP), 28.93 ± 0.48 (CTP) and 22.01 ± 1.48 (UTP), and Vmax (fmol nucleotide/pmol RdRp/min) were 2.40 ± 0.032 (GTP), 1.95 ± 0.17 (ATP), 2.07 ± 0.17 (CTP), and 1.52 ± 0.38