Excision of Incorporated Nucleotide Analogue Chain-terminators can Diminish their Inhibitory Effects on Viral RNA-dependent RNA Polymerases

Abstract

Bovine viral diarrhea virus (BVDV) is amongst the best-characterized members of the Flaviviridae, that includes the hepatitis C virus (HCV). The virally encoded RNA-dependent RNA polymerase (RdRp) plays a crucial role during replication and therefore represents an important target for the development of antiviral drugs. Here we studied biochemical mechanisms associated with the inhibition of BVDV RNA synthesis by 2′-hydroxyl, 3′-deoxynucleoside triphosphates (3′-dNTPs). All four nucleotide analogues are effectively incorporated and act as chain-terminators. However, relatively low, physiologically relevant concentrations of pyrophosphate (PPi) are sufficient to drive the reaction backwards, which results in primer unblocking and rescue of RNA synthesis. Metal ion requirements for nucleotide incorporation and pyrophosphorolysis are similar; the efficiency of both reactions is higher with Mn2+ as compared to Mg2+. Complexes containing chain-terminated primer strands are stable in the presence of heparin, which increases the probability that pyrophosphorolysis occurs before the enzyme can dissociate from its nucleic acid substrate. In contrast to the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT), the BVDV RdRp may not recruit NTP pools as PPi donors. Conversely, we found that the efficiency of primer unblocking is severely compromised in the presence of increasing concentrations of the NTP that is complementary to the next template position. These data suggest that the incoming NTP can access its designated binding site, which, in turn, prevents the catalytically competent complexation of PPi. The results of this study provide novel insights into mechanisms involved in pyrophosphorolysis associated with viral RdRps, and suggest that the excision reaction is likely to be an important parameter that can affect susceptibility to nucleotide analogue inhibitors directed against viral RdRps.