title: 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)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.
authors.affiliation: 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.
authors.forename: K
authors.initials: K
authors.lastname: Barral
authors.forename: C
authors.initials: C
authors.lastname: Sallamand
authors.forename: C
authors.initials: C
authors.lastname: Petzold
authors.forename: B
authors.initials: B
authors.lastname: Coutard
authors.forename: A
authors.initials: A
authors.lastname: Collet
authors.forename: Y
authors.initials: Y
authors.lastname: Thillier
authors.forename: J
authors.initials: J
authors.lastname: Zimmermann
authors.forename: J-J
authors.initials: JJ
authors.lastname: Vasseur
authors.forename: B
authors.initials: B
authors.lastname: Canard
authors.forename: J
authors.initials: J
authors.lastname: Rohayem
authors.forename: F
authors.initials: F
authors.lastname: Debart
authors.forename: E
authors.initials: E
authors.lastname: Decroly
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D003715:Dengue / Q000188:drug therapy / Q000821:virology*; D003716:Dengue Virus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; C582223:NS5 protein, dengue virus; D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases
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