Chen Yongjun, Shi Yujin, Zuo Xiaoyan, Dong Xiaojing, Xiao Xia, Chen Lan, Xiang Zichun, Ren Lili, Zhou Zhuo, Wei Wensheng, Lei Xiaobo, Wang Jianwei
NHC Key Laboratory of System Biology of Pathogens, Christophe Mérieux Laboratory National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
Key Laboratory of Pathogen Infection Prevention and Control (Peking Union Medical College) Ministry of Education, Beijing, China.
J Virol. 2025 Jun 18:e0074125. doi: 10.1128/jvi.00741-25.
Since the outbreak of SARS-CoV-2, viral mutations have posed significant challenges in identifying therapeutic targets and developing broad-spectrum antiviral drugs. Post-translational modifications of genes involved in interferon production and signaling pathways play a crucial role in regulating interferon responses. In this study, we employed CRISPR-Cas9 screening based on adenine base editors to investigate functional amino acids in 1,278 innate immune-related genes. This approach, which converts A-T base pairs into G-C base pairs to probe the functional importance of specific amino acids, allowed us to identify 17 vital factors involved in SARS-CoV-2 infection. Among the candidate genes, genetic knockdown of EHMT2 exhibited the strongest antiviral effect. Further analysis revealed that UNC0638, a selective inhibitor of EHMT2, significantly reduced the endosomal entry of SARS-CoV-2 in pseudovirus assays. The observed inhibitory effect was consistently observed across multiple SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Mechanistically, UNC0638 reduced mature cathepsin L (CTSL) levels, impairing the proteolytic cleavage of SARS-CoV-2 spike protein and subsequent membrane fusion, a critical step for viral entry. Our findings uncover EHMT2 as a host dependency factor and reveal the antiviral mechanism of EHMT2 inhibitors through CTSL maturation blockade. These results advance the understanding of host factors in SARS-CoV-2 infection and provide a strategic framework for developing host-targeted antiviral therapies.IMPORTANCEIn this study, we demonstrated that knockdown or knockout of EHMT2 inhibited SARS-CoV-2 infection, and inhibitors of EHMT2, including UNC0638, UNC0642, and BIX01294 showed similar restrictive effects. Mechanistically, the EHMT2 inhibitor UNC0638 restricts spike-mediated cell entry by inhibiting the maturation of CTSL, a critical protease required for SARS-CoV-2 entry via the endosomal pathway. Importantly, CTSL is not only essential for SARS-CoV-2 but also plays a key role in the entry of other coronaviruses that utilize similar pathways. Therefore, EHMT2 inhibitors could have broader applications as pan-coronavirus therapeutic agents.
自严重急性呼吸综合征冠状病毒2（SARS-CoV-2）爆发以来，病毒突变给确定治疗靶点和开发广谱抗病毒药物带来了重大挑战。参与干扰素产生和信号通路的基因的翻译后修饰在调节干扰素反应中起着关键作用。在本研究中，我们采用基于腺嘌呤碱基编辑器的CRISPR-Cas9筛选方法，研究了1278个先天免疫相关基因中的功能性氨基酸。这种将A-T碱基对转化为G-C碱基对以探究特定氨基酸功能重要性的方法，使我们能够鉴定出17个参与SARS-CoV-2感染的关键因子。在候选基因中，EHMT2的基因敲低表现出最强的抗病毒效果。进一步分析表明，EHMT2的选择性抑制剂UNC0638在假病毒试验中显著降低了SARS-CoV-2的内体进入。在包括阿尔法、贝塔、德尔塔和奥密克戎在内的多个SARS-CoV-2变体中均观察到了这种抑制作用。从机制上讲，UNC0638降低了成熟组织蛋白酶L（CTSL）的水平，损害了SARS-CoV-2刺突蛋白的蛋白水解切割及随后的膜融合，而膜融合是病毒进入的关键步骤。我们的研究结果揭示了EHMT2是一种宿主依赖因子，并通过CTSL成熟阻断揭示了EHMT2抑制剂的抗病毒机制。这些结果加深了对SARS-CoV-2感染中宿主因子的理解，并为开发针对宿主的抗病毒疗法提供了战略框架。重要性在本研究中，我们证明敲低或敲除EHMT2可抑制SARS-CoV-2感染，包括UNC0638、UNC0642和BIX01294在内的EHMT2抑制剂表现出类似的抑制作用。从机制上讲，EHMT2抑制剂UNC0638通过抑制CTSL的成熟来限制刺突介导的细胞进入，CTSL是SARS-CoV-2通过内体途径进入所必需的关键蛋白酶。重要的是，CTSL不仅对SARS-CoV-2至关重要，而且在利用类似途径的其他冠状病毒的进入中也起着关键作用。因此，EHMT2抑制剂作为泛冠状病毒治疗剂可能具有更广泛的应用。
