Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2-4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 μM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.

中文翻译：

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Benzamidine ML336 抑制委内瑞拉马脑炎病毒的正链和负链 RNA 合成，而不影响宿主 RNA 的产生。

委内瑞拉马脑炎病毒（VEEV）是一种美洲流行的甲病毒。VEEV 爆发定期发生，并导致人类和马科动物脑炎。目前尚无用于治疗人类 VEEV 的疗法或疫苗。我们的小组之前曾报道过苯甲脒 VEEV 抑制剂 ML336 的开发，该抑制剂在体外和体内感染模型中均显示出有效的抗病毒活性。在细胞培养实验中，感染后 2-4 小时添加 ML336 会抑制病毒 RNA 合成，并且病毒非结构蛋白（nsP2 和 nsP4）内会发生赋予耐药性的突变。我们假设 ML336 靶向病毒复制酶复合物的活性并抑制病毒 RNA 合成。为了检验这一假设，我们采用了各种生化和细胞测定。使用 ML336 的结构类似物，我们证明这些化合物的细胞抗病毒活性与其对病毒 RNA 合成的抑制相关。例如，ML336 抑制 VEEV RNA 合成的 IC50 测定为 1.1 nM，表明在低纳摩尔范围内具有有效的抗 RNA 合成活性。虽然 ML336 有效抑制 VEEV RNA 合成，但观察到对旧世界甲病毒基孔肯雅病毒 (IC50 > 4 μM) 的作用要弱得多，这与之前基于细胞的测定的数据一致。使用氚掺入测定，我们证明细胞转录没有显着抑制。通过结合荧光成像、链特异性 qRT-PCR 和氚掺入，我们证明 ML336 抑制 VEEV 的正义基因组、负义模板和亚基因组 RNA 的合成。基于这些结果，我们提出此类抗病毒化合物的作用机制是通过与病毒复制酶复合物相互作用来抑制病毒RNA合成。