Pohjala Leena, Barai Vladimir, Azhayev Alex, Lapinjoki Seppo, Ahola Tero
Program in Cellular Biotechnology, Institute of Biotechnology, P.O. Box 56 (Viikinkaari 9), University of Helsinki, 00014 Helsinki, Finland.
Antiviral Res. 2008 Jun;78(3):215-22. doi: 10.1016/j.antiviral.2008.01.001. Epub 2008 Feb 4.
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.
广泛存在的甲病毒属中的几种病毒具有致病性，但目前尚无治疗这些RNA病毒感染的疗法。我们在此报告一种用于筛选塞姆利基森林病毒（SFV）复制抑制剂的定量检测方法，并展示了29种核苷对SFV和辛德毕斯病毒复制的影响。所开发的抗SFV检测方法基于一种在nsP3编码区之后插入了海肾荧光素酶的SFV毒株，产生了一种标记病毒，其中荧光素酶在多蛋白加工过程中被切割出来。该报告基因检测方法被微型化、自动化并经过验证，Z'值为0.52。在病毒RNA合成的最大时间点用[3H]尿苷标记1小时作为一种比较方法。对细胞活力进行抗SFV筛选和反筛选导致发现了几种新的SFV抑制剂。3'-氨基-3'-脱氧腺苷是该组中最有效的抑制剂，在报告基因检测中的IC50值为18 microM，在RNA合成速率检测中的IC50值为2 microM。除了3'-取代类似物外，某些N6-取代核苷对SFV和辛德毕斯病毒复制具有相似的IC50值，这表明该方法总体上适用于甲病毒。
