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Antiviral Res. 2013 Sep;99(3):292-300. doi: 10.1016/j.antiviral.2013.06.001. Epub 2013 Jun 12.

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

Author information

1
AFMB, CNRS, Aix-Marseille Université, UMR 7257, Case 932, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France. Electronic address: karine.barral@afmb.univ-mrs.fr.

Abstract

Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'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, GpppA(2'OMe)-RNA₇₄, by ligation of two RNA fragments. Then, we use GpppA(2'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.

KEYWORDS:

ATA; ATP; AdoHcy; AdoMet; Antiviral drug screening; Cap RNAs synthesis; DEAE; DENV; DF; DHF; DSS; DTT; Dengue virus methyltransferase; EDTA; GTP; HTS; IC(50); MTase; N7-methyltransferase inhibition assay; NS; S-adenosyl-l-homocysteine; S-adenosyl-l-methionine; S-isobutylthio-5′-deoxyadenosine; SD; SDS–PAGE; SIBA; SLA; SPA; TBDMS; TLC; VV; adenosine triphosphate; aurintricarboxylic acid; dengue fever; dengue hemorrhagic fever; dengue shock syndrome; dengue virus; diethylaminoethyl; dithiothreitol; ethylenediaminetetraacetic acid; guanosine triphosphate; high throughput screening; inhibitory concentration that causes 50% reduction in enzyme activity; methyltransferase; non-structural; scintillation proximity assay; sodium dodecylsulfate polyacrylamide gel electrophoresis; standard deviation; stem-loop A; tert-butyldimethylsilyl; thin layer chromatography; vaccinia virus

PMID:
23769894
DOI:
10.1016/j.antiviral.2013.06.001
[Indexed for MEDLINE]

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