Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

冠状病毒作为一类尚无特异性抗病毒药物的新发人畜共患病毒，引发了严重的公共卫生关切。本研究针对包含16671种多样化化合物的化合物库开展抗人类冠状病毒229E（human coronavirus 229E）活性筛选，鉴定出一款命名为K22的抑制剂，其可特异性靶向膜结合型冠状病毒RNA合成过程。K22在病毒侵入后，于病毒生命周期的早期阶段发挥最强抗病毒活性。具体而言，作为冠状病毒复制标志性结构的双膜囊泡（double membrane vesicles, DMVs），其形成在K22处理后受到显著抑制，同时病毒RNA合成几乎完全受阻。对K22产生耐药性的病毒在非结构蛋白6（non-structural protein 6, nsp6）中存在氨基酸替换；而非结构蛋白6是病毒复制复合物的跨膜整合组分，且参与双膜囊泡的形成，这一结果进一步证实K22的作用靶点为膜结合型病毒RNA合成途径。除获得K22耐药性外，nsp6突变株诱导产生的双膜囊泡数量减少，比感染性降低，但其RNA合成并未受到影响。值得注意的是，K22可广谱抑制多种冠状病毒，包括中东呼吸综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV），且在代表人类冠状病毒感染入侵端口的原代人上皮细胞培养体系中实现了高效抑制。综上，本研究提出正链RNA病毒生命周期中的一个进化保守步骤——招募宿主细胞膜用于病毒复制——可作为抗病毒干预的易感且极具成药性的靶点。我们预期这一作用模式可作为开发强效抗病毒药物的范式，用于对抗众多动物及人类病毒感染。