Although the alphavirus Venezuelan equine encephalitis virus (VEEV) has been the cause of multiple outbreaks resulting in extensive human and equine mortality and morbidity, there are currently no anti-VEEV therapeutics available. VEEV pathogenicity is largely dependent on targeting of the viral capsid protein (CP) to the host cell nucleus through the nuclear transporting importin (Imp) α/β1 heterodimer. Here we perform a high-throughput screen, combined with nested counterscreens to identify small molecules able to inhibit the Impα/β1:CP interaction for the first time. Several compounds were able to significantly reduce viral replication in infected cells. Compound G281-1564 in particular could inhibit VEEV replication at low μM concentration, while showing minimal toxicity, with steady state and dynamic quantitative microscopic measurements confirming its ability to inhibit CP nuclear import. This study establishes the principle that inhibitors of CP nucleocytoplasmic trafficking can have potent antiviral activity against VEEV, and represents a platform for future development of safe anti-VEEV compounds with high efficacy and specificity.

尽管阿尔法病毒委内瑞拉马脑炎病毒（VEEV）引起了多次暴发，导致大量人和马死亡和发病，但目前尚无抗VEEV治疗药物。VEEV的致病性主要取决于通过核转运重要蛋白（Imp）α/β1异二聚体将病毒衣壳蛋白（CP）靶向宿主细胞核。在这里，我们进行了高通量筛选，并与嵌套的对位筛选相结合，以鉴定能够首次抑制Impα/β1：CP相互作用的小分子。几种化合物能够显着减少感染细胞中的病毒复制。化合物G281-1564特别可以在低μM浓度下抑制VEEV复制，同时显示出最小的毒性，具有稳定状态和动态定量显微镜测量结果，证实了其抑制CP核进口的能力。这项研究确立了CP核质运输抑制剂可以对VEEV具有有效抗病毒活性的原理，并为将来开发具有安全性和特异性的安全抗VEEV化合物提供了平台。