3′,5′di-O-Trityluridine Inhibits Flavivirus (Dengue and Yellow Fever Virus) Replication and Targets the Viral RNA Dependent RNA Polymerase

Abstract

The dengue fever virus (DENV) and the yellow fever virus (YFV) are members of the genus flavivirus in the family Flaviviridae. An estimated 50-100 million cases of DENV infections occur each year and approximately half a million patients require hospitalization. There is no vaccine or effective antiviral treatment available. There is an urgent need for potent and safe inhibitors of DENV replication; ideally such compounds should have broad-spectrum activity against flaviviruses.We report on the activity of 3',5'di-O-trityluridine (DiTU) on flavivirus replication. The compound inhibits induction of DENV- and YFV-induced cytopathic effect (CPE) with the EC50 values in the low micromolar range. This was confirmed in virus yield reduction experiments, where dose-dependent inhibition of viral RNA synthesis was observed (DENV-2 EC50 = 1.2(mu)M; YFV-17D EC50 = 0.8(mu)M; Selectivity index > 125). Moreover, DiTU also efficiently inhibited viral protein synthesis in the same concentration range. Activity was demonstrated in DENV subgenomic replicons (which encodes only non-structural viral proteins) (EC50 =3'M) indicating that the compoundinhibits intracellular events of the viral replication cycle. This observation was corroborated by the time-of-drug-addition studies, where DiTU was shown to inhibit flavivirus replication at a time point that coincides with the onset of intracellular viral RNA synthesis. DiTU efficiently inhibited highly purified DENV-2 polymerase (EC50 = 1.8(mu)M). Drug-resistant variants are currently being selected, but even following 10 passages, no such variants have so far been selected. In conclusion, our data indicate that the antiflavivirus activity of DiTU is the result of inhibition of the viral RNA dependent RNA polymerase, that this molecule has a high barrier to resistance and that it does not act as a nucleoside analogue.