Developing a robust in vitro assay to measure inhibition of flaviviral RNA polymerases by nucleoside analogues

Abstract

Mosquito borne flaviviruses, such as Dengue virus (DENV) and Zika virus (ZIKV), are globally distributed, and are now endemic in our nearest Pacific neighbours. These viruses infect many millions of people annually, causing serious and sometimes fatal disease. Currently there is only one effective vaccine to prevent the transmission of Yellow Fever virus (YFV), and no specific treatments are available for flaviviral infections. Nucleoside analogues, which can interfere with viral RNA synthesis, are an attractive and well-proven class of small molecule therapeutics. These analogues exploit the general lack of error checking by viral RNA polymerases, and often act by inducing premature chain termination. The recently developed antiviral Galidesivirâ, an iminoribotol-C-nucleoside, is one such compound. Galidesivirâ has been shown to suppress replication of flaviviruses in cell-based assays, and afford protection against infection in some animal models. My research objective was to establish an inhibition assay for the RNA-dependent RNA polymerases (RdRps) of DENV2, DENV3, ZIKV and YFV for testing of Galidesivirâ and related compounds. Flaviviral RdRps were cloned, expressed, purified, and biophysically characterized. Assays were developed to continuously monitor RNA synthesis through the detection of double-stranded RNA, and used to compare basal RdRp activity. These assays are now ready to be deployed to investigate the inhibitory activity of Galidesivirâ and other nucleoside analogues using in vitro approaches.