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  • Title: A luciferase-based screening method for inhibitors of alphavirus replication applied to nucleoside analogues.
    Author: Pohjala L, Barai V, Azhayev A, Lapinjoki S, Ahola T.
    Journal: Antiviral Res; 2008 Jun; 78(3):215-22. PubMed ID: 18294708.
    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.
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