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

Abstract : Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA2'OMe-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-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 (7Me)GpppA-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.
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https://hal.archives-ouvertes.fr/hal-00874640
Contributor : Patrice Heritier Connect in order to contact the contributor
Submitted on : Friday, October 18, 2013 - 3:39:58 PM
Last modification on : Tuesday, December 7, 2021 - 12:32:02 PM

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Karine Barral, Corinne Sallamand, Christiane Petzold, Bruno Coutard, Axelle Collet, et al.. Development of specific dengue virus 2'-O- and N7-methyltransferase assays for antiviral drug screening.. Antiviral Research, Elsevier Masson, 2013, 99 (3), pp.292-300. ⟨10.1016/j.antiviral.2013.06.001⟩. ⟨hal-00874640⟩

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