title: A luciferase-based screening method for inhibitors of alphavirus replication applied to nucleoside analogues.
abstract: Several members of the widespread alphavirus group are pathogenic, but no therapy is available to treat these RNA virus infections. We report here a quantitative assay to screen for inhibitors of Semliki Forest virus (SFV) replication, and demonstrate the effects of 29 nucleosides on SFV and Sindbis virus replication. The anti-SFV assay developed is based on a SFV strain containing Renilla luciferase inserted after the nsP3 coding region, yielding a marker virus in which the luciferase is cleaved out during polyprotein processing. The reporter-gene assay was miniaturized, automated and validated, resulting in a Z' value of 0.52. [3H]uridine labeling for 1 h at the maximal viral RNA synthesis time point was used as a comparative method. Anti-SFV screening and counter-screening for cell viability led to the discovery of several new SFV inhibitors. 3'-amino-3'-deoxyadenosine was the most potent inhibitor in this set, with an IC50 value of 18 microM in the reporter-gene assay and 2 microM in RNA synthesis rate detection. Besides the 3'-substituted analogues, certain N6-substituted nucleosides had similar IC50 values for both SFV and Sindbis replication, suggesting the applicability of this methodology to alphaviruses in general.
authors.affiliation: Program in Cellular Biotechnology, Institute of Biotechnology, P.O. Box 56 (Viikinkaari 9), University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Leena
authors.initials: L
authors.lastname: Pohjala
authors.forename: Vladimir
authors.initials: V
authors.lastname: Barai
authors.forename: Alex
authors.initials: A
authors.lastname: Azhayev
authors.forename: Seppo
authors.initials: S
authors.lastname: Lapinjoki
authors.forename: Tero
authors.initials: T
authors.lastname: Ahola
mesh_terms: D000524:Alphavirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D003839:Deoxyadenosines / Q000737:chemistry / Q000494:pharmacology; D017930:Genes, Reporter; D020128:Inhibitory Concentration 50; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests / Q000379:methods; D009705:Nucleosides / Q000737:chemistry / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis; D012672:Semliki forest virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D012845:Sindbis Virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D003839:Deoxyadenosines; D009705:Nucleosides; D012367:RNA, Viral; C019247:3'-amino-3'-deoxyadenosine; D008156:Luciferases