Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.
Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.
复制标题
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。
DOI：
10.1016/j.antiviral.2019.104674
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发表时间：
2020
影响因子：
7.6
通讯作者：
Chung,Dong-Hoon
中科院分区：
文献类型：
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作者：
Skidmore,AndrewM;Adcock,RobertS;Jonsson,ColleenB;Golden,JenniferE;Chung,Dong-Hoon
委内瑞拉马脑炎病毒(VEEV)是美洲特有的一种甲型病毒。Veev疫情周期性发生，在人类和马类中都会引起脑炎。目前还没有治疗人类VEEV的疗法或疫苗。我们小组此前曾报道开发出一种苯并嘧啶VEEV抑制剂ML336，它在体外和活体感染模型中显示出强大的抗病毒活性。在细胞培养实验中，ML336在感染后2-4小时加入时会抑制病毒RNA的合成，并在病毒非结构蛋白(nsP2和nsp4)内发生导致耐药性的突变。我们假设ML336针对病毒复制酶复合体的活性，并抑制病毒RNA的合成。为了验证这一假设，我们采用了各种生化和细胞分析方法。利用ML336的结构类似物，我们证明了这些化合物的细胞抗病毒活性与它们对病毒RNA合成的抑制有关。例如，ML336对VEEV RNA合成的抑制IC50为1.1 nM，表明在低纳摩尔范围内有很强的抗RNA合成活性。虽然ML336有效地抑制了VEEV的RNA合成，但对旧大陆甲型病毒基孔肯雅病毒(IC50>；F4和μM)的作用要弱得多，这与之前基于细胞的检测数据一致。使用氚掺入实验，我们证明了没有明显的细胞转录抑制。结合荧光、链特异性qRT-PCR和氚掺入，我们证明ML336抑制VEEV正义基因组、负义模板和亚基因组RNA的合成。基于这些结果，我们认为这类抗病毒化合物的作用机制是通过与病毒复制酶复合体相互作用来抑制病毒RNA的合成。
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 bothin vitroandin vivomodels 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 IC50of 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.