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 IC 50 value of 18 μM in the reporter-gene assay and 2 μM in RNA synthesis rate detection. Besides the 3′-substituted analogues, certain N 6-substituted nucleosides had similar IC 50 values for both SFV and Sindbis replication, suggesting the applicability of this methodology to alphaviruses in general.
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