Our previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection. In this study, we aimed to design and construct a bivalent protein consisting of GRFT and EK1 components and evaluate its inhibitory activity and mechanism of action against infection by SARS-CoV-2 and its mutants, as well as other human coronaviruses (HCoVs). The bivalent proteins were expressed in E. coli and purified with Ni-NTA column. HIV backbone-based pseudovirus (PsV) infection and HCoV S-mediated cell–cell fusion assays were performed to test their inhibitory activity. ELISA and Native-PAGE were conducted to illustrate the mechanism of action of these bivalent proteins. Five-day-old newborn mice were intranasally administrated with a selected bivalent protein before or after HCoV-OC43 challenge, and its protective effect was monitored for 14 days. Among the three bivalent proteins purified, GL25E exhibited the most potent inhibitory activity against infection of SARS-CoV-2 PsVs expressing wild-type and mutated S protein. GL25E was significantly more effective than GRFT and EK1 alone in inhibiting HCoV S-mediated cell–cell fusion, as well as infection by SARS-CoV-2 and other HCoVs, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. GL25E could inhibit authentic SASR-CoV-2, HCoV-OC43 and HCoV-229E infection in vitro and prevent newborn mice from authentic HCoV-OC43 infection in vivo. GL25E could bind to glycans in the S1 subunit and HR1 in the S2 subunit in S protein, showing a mechanism of action similar to that of GRFT and EK1 alone. Since GL25E showed highly potent and broad-spectrum inhibitory activity against infection of SARS-CoV-2 and its mutants, as well as other HCoVs, it is a promising candidate for further development as a broad-spectrum anti-HCoV therapeutic and prophylactic to treat and prevent COVID-19 and other emerging HCoV diseases.

中文翻译：

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刺突蛋白中靶向聚糖和 HR1 结构域的二价蛋白可有效抑制 SARS-CoV-2 和其他人类冠状病毒的感染

我们之前的研究表明，将抗病毒凝集素 GRFT 和 pan-CoV 融合抑制肽 EK1 相结合，可以对 SARS-CoV-2 感染产生高效的抑制活性。在本研究中，我们旨在设计和构建由 GRFT 和 EK1 成分组成的二价蛋白，并评估其对 SARS-CoV-2 及其突变体以及其他人类冠状病毒 (HCoV) 感染的抑制活性和作用机制。二价蛋白在大肠杆菌中表达并用 Ni-NTA 柱纯化。进行了基于 HIV 骨架的假病毒 (PsV) 感染和 HCoV S 介导的细胞 - 细胞融合试验以测试它们的抑制活性。进行ELISA和Native-PAGE以说明这些二价蛋白质的作用机制。五天大的新生小鼠在 HCoV-OC43 攻击之前或之后鼻内给予选定的二价蛋白，并监测其保护作用 14 天。在纯化的三种二价蛋白中，GL25E 对表达野生型和突变型 S 蛋白的 SARS-CoV-2 PsV 的感染表现出最有效的抑制活性。GL25E 在抑制 HCoV S 介导的细胞-细胞融合以及 SARS-CoV-2 和其他 HCoV（包括 SARS-CoV、MERS-CoV、HCoV-229E、HCoV- NL63 和 HCoV-OC43。GL25E可以在体外抑制真正的SASR-CoV-2、HCoV-OC43和HCoV-229E感染，并在体内防止新生小鼠受到真正的HCoV-OC43感染。GL25E 可以与 S 蛋白中 S1 亚基中的聚糖和 S2 亚基中的 HR1 结合，显示出类似于单独的 GRFT 和 EK1 的作用机制。由于 GL25E 对 SARS-CoV-2 及其突变体以及其他 HCoV 的感染显示出高效的广谱抑制活性，因此它是进一步开发作为广谱抗 HCoV 治疗和预防治疗的有希望的候选者并预防 COVID-19 和其他新出现的 HCoV 疾病。