Detection and quantification of flavivirus NS5 methyl-transferase activities

Siew Pheng Lim, Christophe Bodenreider, Pei Yong Shi

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Scopus citations

Abstract

Flavivirus NS5 is the most conserved protein amongst the flavivirus proteins and is an essential enzyme for viral mRNA capping and replication. It encodes a methyl-transferase (MTase) domain at its N-terminal region which carries out sequential N7 and 2′-O methylation, resulting in the formation of the cap1 structure on its viral RNA genome. Two key methods have been established to measure these activities in vitro: thin-layer chromatography (TLC) and scintillation proximity assays (SPA). TLC offers the advantage of direct visualization of the amounts and types of cap structures formed whilst the SPA assay is more sensitive and quantitative. It is also amenable to high-throughput compound screening. The drawback of both assays is the need for radioisotope usage. We further describe the adaptation of a nonradioactive immune-competitive fluorescence polarization assay for detection of dengue virus MTase activity.

Original languageEnglish (US)
Title of host publicationAntiviral Methods and Protocols
Pages249-268
Number of pages20
DOIs
StatePublished - Jul 18 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1030
ISSN (Print)1064-3745

Keywords

  • Dengue virus
  • Flavivirus
  • Fluorescence polarization assay
  • Methyl-transferase
  • NS5
  • RNA capping
  • Scintillation proximity assay
  • Thin-layer chromatography

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Lim, S. P., Bodenreider, C., & Shi, P. Y. (2013). Detection and quantification of flavivirus NS5 methyl-transferase activities. In Antiviral Methods and Protocols (pp. 249-268). (Methods in Molecular Biology; Vol. 1030). https://doi.org/10.1007/978-1-62703-484-5_20