新型冠状病毒（Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2）感染引起的新型冠状病毒肺炎（Coronavirus Disease 2019, COVID-19）的全球大流行，对世界各国人民的健康和经济造成了巨大的威胁，但目前对SARS-CoV-2的致病机制的研究仍不十分清楚，揭示其致病机制对于COVID-19的防控具有重要意义。

天然免疫是宿主抵抗病毒感染的第一道防线，在抵抗病原微生物感染的过程中发挥重要作用。病毒入侵后，被宿主细胞模式识别受体识别，活化并诱导一系列级联反应，激活宿主细胞天然免疫信号通路，诱导干扰素等细胞因子的产生。本团队的前期研究表明I型干扰素（Interferon-I, IFN-I）可显著抑制SARS-CoV-2的感染。有报道证明，携带天然免疫反应关键分子的基因突变的患者中，重症风险显著提高。另外，患者体内存在干扰素自身抗体，也是导致COVID-19患者发展为重症的关键因素之一。目前对于宿主细胞抵抗SARS-CoV-2感染的天然免疫反应及关键分子仍缺乏全面的了解。

天然免疫信号通路的关键分子，在信号通路活化及恢复静息状态的过程中，受到精密的调控，这些迅速而具有时效性的反应大多通过调控蛋白质的翻译后修饰(Posttranslational modification, PTM)来实现，如磷酸化、泛素化、乙酰化和苏素化，大多数翻译后修饰主要发生在赖氨酸（Lysine, K）、丝氨酸（Serine, S ）、苏氨酸（Threonine, T）、酪氨酸（Tyrosine, Y）上，这些氨基酸的共同特点是其侧链基团都有氨基或羟基，可以在其侧链上添加官能团来进行各种翻译后修饰。这些蛋白修饰位点的氨基酸改变，会导致蛋白修饰的消失，从而失去对相应蛋白功能的调控。本研究针对天然免疫信号通路的1278个关键分子，构建了含有近八万条小向导RNA（Small guide RNA, sgRNA）的Cas9文库，导入A549-ACE2-ABE（adenine base editor）细胞（融合表达dCas9与腺嘌呤脱氨酶），文库覆盖此1278个关键蛋白所有K, S, T, Y对应的基因组腺苷酸位点，通过ABE-CRISPR dCas9实现碱基的转换，转录翻译后，实现蛋白相应氨基酸位点的突变，从而实现蛋白质翻译后修饰方式的改变。文库还包含针对血管紧张素转化酶2（Angiotensin converting enzyme2, ACE2）的sgRNA30条，阴性对照sgRNA 500条。针对得到的细胞库，在生物安全三级实验室（The Biological safety level-3 Laboratory, BSL-3）进行SARS-CoV-2感染，经过四轮感染后，对存活细胞进行测序，从而得到存活细胞所携带的突变类型信息。两次独立重复实验共获得51个候选分子的74个氨基酸突变位点。突变后细胞存活，说明细胞不易被SARS-CoV-2感染，提示细胞携带的突变导致病毒不易感染宿主细胞。

对筛选到的阳性对照ACE2的氨基酸突变位点进行验证，发现这些位点突变均引起ACE2蛋白表达的降低，ACE2为SARS-CoV-2入侵细胞的受体，其表达的降低显著抑制了病毒的感染，导致细胞存活，提示本筛选体系有效，可以获得抑制病毒复制的sgRNA。筛选到的51个候选分子中，常染色体赖氨酸组蛋白甲基转移酶2（Euchromatic histone lysine methyltransferase 2, EHMT2）共有五条sgRNA被测序获得，定位于五个不同的氨基酸位点（S872, S895, Y1154, Y1043, Y1097），提示EHMT2可能在病毒感染复制中发挥重要作用。

EHMT2为常染色质组蛋白赖氨酸甲基转移酶，有报道证明其催化组蛋白H3的9位的赖氨酸发生单甲基化和二甲基化，导致转录被抑制。最近研究表明EHMT2除影响组蛋白的甲基化外，也影响非蛋白分子的甲基化，进而影响其功能。EHMT2在许多癌症细胞中高表达，其抑制剂为潜在的抗癌药物，BIX-01294是最早发现的EHMT2的特异性抑制剂。为了验证EHMT2在SARS-CoV-2感染中的作用，我们首先验证EHMT2抑制剂对于病毒复制的影响，发现在Vero细胞和A549细胞中使用BIX-01294预处理均能够显著抑制SARS-CoV-2病毒的感染进程，其IC50分别为0.98 μM和0.24 μM。Western Blot结果也显示抑制剂处理显著降低了新冠病毒的棘突（Spike, S）蛋白和核衣壳（Nucleocapsid, N）蛋白的表达。此外，另外两种EHMT2特异性抑制剂UNC0638和UNC0642也表现出对SARS-CoV-2感染的显著抑制作用。使用siRNA对EHMT2的表达进行敲低也明显抑制SARS-CoV-2的感染。同时BIX-01294处理以及EHMT2表达的敲低也可抑制流感病毒等RNA病毒的感染，提示EHMT2可能是一个广谱的抗病毒靶标。

文献报道表明，EHMT2的抑制可导致I型干扰素表达的上升，但是我们在BIX-01294处理后并未发现其介导的I型干扰素的表达上升，且在干扰素基因缺失的Vero细胞和STAT1敲除的A549细胞中BIX-01294同样可以抑制SARS-CoV-2的复制，提示其抗病毒作用并不依赖于I型干扰素，其具体作用机制仍需进一步的研究。

The global pandemic of New Coronavirus pneumonia (COVID-19) caused by New Coronavirus (SARS-CoV-2) has posed a great threat to the health and economy of people all over the world. But at present, the understanding of the pathogenic mechanism of SARS-CoV-2 is not very clear. Revealing its pathogenic mechanism is of great significance for the prevention and control of COVID-19.

Innate immunity is the first line of defense for the host to resist viral infection and plays an important role in the process of resisting infection. After the virus invaded, it is recognized by the host cell pattern recognition receptor, activated and induced a series of cascade reactions, activated the host cell innate immune signaling pathway, induced the production of interferon and other cytokines. Previous studies by our team have shown that interferon-I (IFN-I) can significantly inhibit SARS-COV-2 infection. It has been reported that patients with genetic mutations that carry molecules critical to the innate immune response have a significantly increased risk of severe illness. In addition, the presence of interferon autoantibodies in patients is also one of the key factors leading to the development of severe disease in COVID-19 patients. At present, the innate immune response and key molecules of host cells against SARS-COV-2 infection are still not fully understood.

Key molecules of the innate immune signaling pathway are precisely regulated during the activation and restoration of the signaling pathway to its resting state. These rapid and time-sensitive reactions are mostly achieved by regulating Posttranslational modification (PTM) of proteins, such as phosphorylated, ubiquitin, acetylation and sutenylation. Most post-translational modifications mainly occur in Lysine (K), Serine (S), Threonine (T) and Tyrosine (Y). The common feature of these amino acids is that their side chain groups all have amino or hydroxyl groups, and functional groups can be added to their side chains for various post-translational modifications. The amino acid changes of these protein modification sites will lead to the disappearance of protein modification, thus losing the regulation of the corresponding protein functions. In this study, aiming at 1278 key molecules of innate immune signaling pathway, Cas9 library containing nearly 80,000 small guide RNA (sgRNA) was constructed and introduced into A549-ACE2-ABE (Adenine Base Editor) cells (dCas9 and adenine deaminase were fused and expressed). The library covers all the genomic adenylate sites corresponding to K, S, T and Y of these 1278 key proteins, and realizes base conversion through ABE-CRISPR dCas9. After transcription and translation, the corresponding amino acid sites of the protein are mutated, thus the modification mode of the protein after translation is changed. The library also contains 30 sgRNA targeting Angiotensin converting enzyme2 (ACE2) and 500 sgRNA negative controls. The obtained cell bank was infected with SARS-CoV-2 in the biological safety level-3 laboratory (BSL-3). After four rounds of infection, the surviving cells were sequenced, and the mutation information carried by the surviving cells was obtained. A total of 74 amino acid mutation sites of 51 candidate molecules were obtained by two independent experiments. After mutation, the cells survived, indicating that the cells were not easily infected by SARS-CoV-2, suggesting that the mutations carried by the cells made it difficult for the virus to infect the host cells.

We verified the amino acid mutation sites of ACE2, a positive control. We found that mutations decreased the expression of ACE2 protein. ACE2 is the receptor of SARS-COV-2 invading cells. The decrease of its expression significantly inhibited virus infection, leading to cell survival. It is suggested that this screening system is effective and can obtain sgRNA which can inhibit virus replication. Among 51 candidate molecules, five sgRNA of euchromosome lysine methyl transferase 2 (EHMT2) were sequenced and located at 5 different amino acid sites(S872, S895, Y1154, Y1043, Y1097), suggesting that EHMT2 may play an important role in SARS-CoV-2 infection and replication.

EHMT2 is euchromatin histone lysine methyltransferase. It has been reported that it catalyzes the monomethylation and dimethylization of lysine at position 9 of histone H3, resulting in the inhibition of transcription. Recent studies have shown that EHMT2 not only affects the methylation of histones, but also affects the methylation of non-protein molecules, thus affecting their functions. EHMT2 is highly expressed in many cancer cells. Its inhibitor is a potential anticancer drug. Bix-01294 is the earliest specific inhibitor of EHMT2. To verify the role of EHMT2 in SARS-COV-2 infection, we first validated the effect of EHMT2 inhibitors on viral replication. We found that pretreatment of BIX-01294 in Vero and A549 cells could significantly inhibit the infection process of SARS-CoV-2, with IC50 of 0.98μM and 0.24μM respectively. Western Blot results also showed that inhibitor treatment significantly decreased Spike (S) and Nucleocapsid (N) protein expression in SARS-CoV-2. In addition, two other EHMT2 specific inhibitors UNC0638 and UNC0642 also showed significant inhibitory effects on SARS-CoV-2 infection. Knock-down of EHMT2 expression by siRNA also significantly inhibited SARS-COV-2 infection. At the same time, BIX-01294 and knock-down of EHMT2 expression can also inhibit the infection of RNA viruses such as influenza virus, suggesting that EHMT2 may be a broad-spectrum antiviral target.

Literature reports show that the inhibition of EHMT2 may lead to the rise of type I interferon, but we did not find the rise of type I interferon mediated by BIX-01294 after treatment, and BIX-01294 can inhibit the replication of SARS-CoV-2 in Vero cells with interferon gene deletion and A549 cells with STAT1 knockout, suggesting that its antiviral effect does not depend on type I interferon, and its specific mechanism needs to be further studied.