Zika virus, a member of the Flaviviridae family, has garnered global attention due to the link of recent outbreaks with increased risk of fetal microcephaly and Guillan-Barré syndrome in adults. As of yet, there are no approved therapies for treatment or prevention of Zika virus infection. In this research, we analyze inhibition of Zika virus replication by nucleoside analogs – a therapeutic strategy that has been successful levied against other members of the Flaviviridae family such as Hepatitis C. We have recombinantly expressed and purified the RNA-dependent RNA polymerase (RdRP) domain of the Zika NS5 protein. The purified protein is used in an in vitro primer extension assay to characterize incorporation of native nucleotides and inhibitory analogs opposite a Zika specific template. We find that 2’-C-MeUTP has higher rates of incorporation by Zika RdRP compared to 2’-F- 2’-C-MeUTP. The later analog is the active metabolite of the FDA approved drug sofosbuvir, which is active against Hepatitis C, a virus of the same family. Our comparison of Zika RdRP catalyzed incorporation rates of various nucleotide analogs will enable identification of structural features that drive high affinity for Flavivirus RdRPs. Identification of structural trends will inform future drug design.
