Development of specific dengue virus 2′-O- and N7-methyltransferase assays for antiviral drug screening

•Production of two 74-mer capped RNA substrates, GpppA2′OMe-RNA74 and 7MeGpppA-RNA74, for DENV N7- and 2′-O-MTase.•Development of a robust, specific and high-throughput N7-MTase inhibition assay.•Selective screening of known MTase inhibitors against DENV 2′-O- and N7-MTase.

Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, 7MeGpppA2′OMe-RNA, at the 5′-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, 7MeGpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA2′OMe-RNA74, by ligation of two RNA fragments. Then, we use GpppA2′OMe-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate 7MeGpppA-RNA74 in order to characterize the DENV 2′-O-MTase activity specifically on long capped RNA.We next compared the N7- and 2′-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.