Substrate selectivity of Dengue and Zika virus NS5 polymerase towards 2′-modified nucleotide analogues

•Assembly of stable elongation complexes of dengue and Zika virus proteins NS5 bearing the RdRp activity.•Metal ion dependence of complex assembly and fast NTP incorporation.•Determination of 2′ NTP analogue selectivity and mode of action as non-obligate chain terminators.

In targeting the essential viral RNA-dependent RNA-polymerase (RdRp), nucleotide analogues play a major role in antiviral therapies. In the Flaviviridae family, the hepatitis C virus (HCV) can be eradicated from chronically infected patients using a combination of drugs which generally include the 2′-modified uridine analogue Sofosbuvir, delivered as nucleotide prodrug. Dengue and Zika viruses are emerging flaviviruses whose RdRp is closely related to that of HCV, yet no nucleoside drug has been clinically approved for these acute infections. We have purified dengue and Zika virus full-length NS5, the viral RdRps, and used them to assemble a stable binary complex made of NS5 and virus-specific RNA primer/templates. The complex was used to assess the selectivity of NS5 towards nucleotide analogues bearing modifications at the 2′-position. We show that dengue and Zika virus RdRps exhibit the same discrimination pattern: 2′-O-Me > 2′-C-Me-2′-F > 2′-C-Me nucleoside analogues, unlike HCV RdRp for which the presence of the 2′-F is beneficial rendering the discrimination pattern 2′-O-Me > 2′-C-Me ≥ 2′-C-Me-2′-F. Both 2′-C-Me and 2′-C-Me-2′-F analogues act as non-obligate RNA chain terminators. The dengue and Zika NS5 nucleotide selectivity towards 2′-modified NTPs mirrors potency of the corresponding analogues in infected cell cultures.