Inhibition of influenza virus transcription by 2'-deoxy-2'-fluoroguanosine

Abstract

The nucleoside analog 2-deoxy-2-fluoroguanosine (2-fluorodGuo) is phosphorylated by cellular enzymes and reversibly inhibits influenza virus replication in chick embryo cells within the first 4 h of infection. RNA hybridization studies revealed that primary and secondary transcription of influenza virus RNA were blocked at a compound concentration of 10 M, but no inhibition of cell protein synthesis was seen even at high compound concentrations (200 M). In vitro, the triphosphate of 2-fluorodGuo is a competitive inhibitor of influenza virus transcriptase activity from disrupted virus, with a K i of 1.0 M. The cellular polymerases DNA polymerase ␣ and RNA polymerase II were only weakly inhibited or were insusceptible to 2-fluorodGTP. In kinetic studies with the influenza virus transcriptase, 2-fluorodGTP, in the absence of GTP, blocked elongation of the virus RNA chain. Similarly, by using purified ribonucleoprotein complexes it was found that the addition of a single nucleotide of 2-fluorodGTP to the virus RNA caused chain termination, which resulted in the blockage of further virus transcription. Furthermore, the specificity for influenza virus transcriptase was confirmed when the transcriptase from partially resistant virus was found to be 10-fold less susceptible to 2-fluorodGTP (K i ‫؍‬ 13.1 M).The 2Ј-fluororibosides are potent inhibitors of influenza virus A and B strains in cell culture, in the mouse pneumonia model (16), and in the ferret model (6). All of the influenza virus strains examined were susceptible to 2Ј-deoxy-2Ј-fluoroguanosine (2Ј-fluorodGuo), but the level of inhibition varies with cell type and appears to be related to the levels of phosphorylation in these different cells. In human tracheal cells, growth of influenza virus A/England/40/83 (H3N2) was reduced by 4 log 10 units, suggesting that 2Ј-fluorodGuo is phosphorylated very efficiently in the human target cell. Furthermore, evaluation in human respiratory epithelial explants showed that 2Ј-fluorodGuo was extremely potent against both influenza virus A and B strains (90% effective concentration, Ͻ 0.1 g/ml [Ͻ0.35 M]), and no toxicity was observed at 100 g/ml (350 M) (15). These results indicate that the compound may be particularly effective when it is delivered directly to the respiratory tract.Deoxycytidine kinase is one enzyme which has been shown to be able to phosphorylate both pyrimidine and purine analogs to their 5Ј-monophosphates (17). Furthermore, 2Ј-fluorodGuo monophosphate is a substrate for GMP kinase (K m , 53 M, compared with a K m of 7 to 9 M for GMP [11a]). Correlations between intracellular 2Ј-fluorodGuo triphosphate (2Ј-fluorodGTP) levels and influenza virus inhibition suggest that the compound may be targeting influenza virus RNA replication possibly by inhibiting the influenza virus polymerase complex.The present study was undertaken to elucidate further the mechanism of inhibition of influenza virus replication by 2Ј-fluorodGuo. The triphosphate of 2Ј-fluorodGuo was synthesized for enzyme kinetic studies, including...

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