Quantitative substrate profiling of Zika NS5 methyltransferase activity

Zika virus (ZIKV) is a mosquito-borne and sexually transmitted flavivirus associated with severe congenital abnormalities and neurological complications. Despite its significant global health impact, there are currently no approved antiviral therapies that specifically target ZIKV. Its replication relies on the multifunctional non-structural protein 5 (NS5), whose N-terminal methyltransferase (MTase) domain catalyzes RNA cap methylation, a critical process required for mRNA stability, evasion of the host innate immune responses, and efficient replication. Flavivirus NS5 MTase substrate specificities have been extensively investigated. However, limited quantitative kinetic evaluation is available to compare substrate selectivity. Here we report a quantitative assessment of the substrate preference and catalytic efficiency of ZIKV NS5 MTase activity using a panel of short (ten transcribed nucleotides) synthetic cap RNA substrates with defined cap methylation patterns. Kinetic parameters for both RNA substrates and S-adenosylmethionine were determined using an optimized radiometric assay for characterizing active substrates. The methylation outcomes were further analyzed and validated by liquid chromatography-mass spectrometry. NS5 activity required unmethylated 2'-O position of the first transcribed nucleotide, while pre-methylation of cap N⁷-guanine had little effect on its activity. However, the methylation of the second nucleotide reduced its activity substantially. Our data indicate that NS5 selectively catalyzes 2'-O methylation of the first transcribed nucleotide, with no N⁷-guanine methylation under our experimental conditions. Such methylation site selectivity is similar to that of human CMTR1 in our previous study, necessitating counter-screening of NS5 inhibitors against human CMTR1.