Viral interferon (IFN) antagonists are a diverse class of viral proteins that counteract the host IFN response, which is important for controlling viral infections. Viral IFN antagonists are often multifunctional proteins that perform vital roles in virus replication beyond IFN antagonism. The critical importance of viral IFN antagonists is highlighted by the fact that almost all viruses encode one of these proteins. Inhibition of viral IFN antagonists has the potential to exert pleiotropic antiviral effects and thus this important protein class represents a diverse plethora of novel therapeutic targets. To exploit this, we have successfully developed and executed a novel modular cell-based platform that facilitates the safe and rapid screening for inhibitors of a viral IFN antagonist of choice. The platform is based on two reporter cell-lines that provide a simple method to detect activation of IFN induction or signaling via an eGFP gene placed under the control of the IFNβ or an ISRE-containing promoter, respectively. Expression of a target IFN antagonist in the appropriate reporter cell-line will block the IFN response and hence eGFP expression. We hypothesized that addition of a compound that inhibits IFN antagonist function will release the block imposed on the IFN response and hence restore eGFP expression, providing a measurable parameter for high throughput screening (HTS). We demonstrate assay proof-of-concept by (i) exploiting hepatitis C virus (HCV) protease inhibitors to inhibit NS3-4A's capacity to block IFN induction and (ii) successfully executing two HTS targeting viral IFN antagonists that block IFN signaling; NS2 and IE1 from human respiratory syncytial virus (RSV) and cytomegalovirus (CMV) respectively, two clinically important viruses for which vaccine development has thus far been unsuccessful and new antivirals are required. Both screens performed robustly and Z′ Factor scores of >0.6 were achieved. We identified (i) four hit compounds that specifically inhibit RSV NS2's ability to block IFN signaling by mediating STAT2 degradation and exhibit modest antiviral activity and (ii) two hit compounds that interfere with IE1 transcription and significantly impair CMV replication. Overall, we demonstrate assay proof-of-concept as we target viral IFN antagonists from unrelated viruses and demonstrate its suitability for HTS.

病毒干扰素 (IFN) 拮抗剂是多种病毒蛋白，可抵消宿主 IFN 反应，这对于控制病毒感染非常重要。病毒干扰素拮抗剂通常是多功能蛋白质，除了干扰素拮抗作用外，它们在病毒复制中发挥重要作用。几乎所有病毒都编码其中一种蛋白质，这一事实凸显了病毒干扰素拮抗剂的至关重要性。病毒干扰素拮抗剂的抑制有可能发挥多效抗病毒作用，因此这一重要的蛋白质类别代表了多种新型治疗靶点。为了利用这一点，我们成功开发并执行了一种新型模块化细胞平台，该平台有助于安全、快速筛选所选病毒干扰素拮抗剂的抑制剂。该平台基于两种报告细胞系，提供了一种简单的方法来检测 IFN 诱导或通过分别置于 IFNβ 或包含 ISRE 的启动子控制下的 eGFP 基因进行信号传导的激活。目标 IFN 拮抗剂在适当的报告细胞系中的表达将阻断 IFN 反应，从而阻断 eGFP 表达。我们假设添加抑制 IFN 拮抗剂功能的化合物将释放对 IFN 反应的阻碍，从而恢复 eGFP 表达，为高通量筛选 (HTS) 提供可测量的参数。 我们通过（i）利用丙型肝炎病毒（HCV）蛋白酶抑制剂抑制 NS3-4A 阻断 IFN 诱导的能力，以及（ii）成功执行两种针对病毒 IFN 拮抗剂的 HTS 来阻断 IFN 信号转导，从而证明了测定概念验证； NS2 和 IE1 分别来自人呼吸道合胞病毒 (RSV) 和巨细胞病毒 (CMV)，这两种临床上重要的病毒的疫苗开发迄今尚未成功，需要新的抗病毒药物。两种屏幕均表现强劲，Z' 因子得分达到 >0.6。我们鉴定了（i）四种命中化合物，它们特异性抑制 RSV NS2 通过介导 STAT2 降解来阻断 IFN 信号传导的能力，并表现出适度的抗病毒活性；（ii）两种命中化合物，干扰 IE1 转录并显着损害 CMV 复制。总体而言，当我们针对不相关病毒的病毒 IFN 拮抗剂时，我们展示了测定概念验证，并证明了其对 HTS 的适用性。