Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: Implication for viral RNA synthesis

Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3 ' end of viral RNAs. However, this T NTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inhere nt function of HCV and bovine viral diarrhea virus RdRps highly purified fr om both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activi ty are strongly influenced by the sequence near the 3 ' terminus of the vir al template RNA, perhaps accounting for the previous discrepant observation s between RdRp preparations. Last, the RdRp TNTase activity was shown to re store the ability to direct initiation of RNA synthesis in vitro on an init iation-defective RNA substrate, thereby implicating this activity in mainta ining the integrity of the viral genome termini.