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The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants that can evade the immunity in a host achieved through vaccination have emerged. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread, are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner and is TMPRSS2 independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific small interfering RNAS (siRNAs) revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytium formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosomal pathways could be an effective strategy by which to cure COVID-19 in the future. To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2. SARS-CoV-2 binds to the cell surface receptor ACE2 via the spike protein, and then the spike protein is cleaved by host proteases to enable entry. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection in addition to the TMPRSS2-mediated pathway and the endosomal pathway. The metalloproteinase-mediated pathway requires both the prior cleavage of spike into two domains and a specific sequence in the second domain, S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. Besides the contribution of metalloproteinases to SARS-CoV-2 infection, inhibition of metalloproteinases was important in preventing cell death, which may cause organ damage. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

全球范围内正在进行预防 SARS-CoV-2 感染（COVID-19 的病原体）的疫苗接种计划已取得显著成功。然而，最近出现了能够逃避宿主通过疫苗接种获得的免疫的病毒变体。因此，迫切需要新的治疗药物来有效预防这些新变体的感染，从而防止 COVID-19 的传播。为此，需要广泛研究病毒-宿主细胞相互作用，以确定有效的治疗靶标。在这里，我们报告了 SARS-CoV-2 的一种细胞表面进入途径，该途径以细胞类型依赖性方式存在，并且不依赖于 TMPRSS2，但对各种广谱金属蛋白酶抑制剂（如 marimastat 和 prinomastat）敏感。使用选择性金属蛋白酶抑制剂和基因特异性小干扰 RNA（siRNA）的实验表明，解整合素金属蛋白酶 10（ADAM10）部分参与了金属蛋白酶途径。与我们的发现一致，即该途径在高致病性人类冠状病毒中是 SARS-CoV-2 所特有的，S1/S2 边界中的弗林裂解基序和 SARS-CoV-2 刺突蛋白的 S2 结构域对于金属蛋白酶依赖性进入都是必不可少的。相比之下，SARS-CoV-2 的这两个元素独立地有助于 TMPRSS2 依赖性 S2 引发。金属蛋白酶途径参与 SARS-CoV-2 诱导的合胞体形成和细胞病变作用，这使我们推断它也参与了 SARS-CoV-2 的快速传播和 COVID-19 的发病机制。因此，除了 TMPRSS2 和内体途径之外，靶向金属蛋白酶途径可能是未来治愈 COVID-19 的有效策略。

为了开发针对 COVID-19 的有效治疗方法，有必要详细阐明病原体 SARS-CoV-2 的感染机制。SARS-CoV-2 通过刺突蛋白与细胞表面受体 ACE2 结合，然后刺突蛋白被宿主蛋白酶切割，从而实现进入。在这里，我们发现除了 TMPRSS2 介导的途径和内体途径之外，金属蛋白酶介导的途径对 SARS-CoV-2 感染很重要。金属蛋白酶介导的途径需要先将刺突蛋白切割成两个结构域，并且第二个结构域 S2 中存在特定序列，这些条件是 SARS-CoV-2 所具备的，但相关的人类冠状病毒 SARS-CoV 却没有。除了金属蛋白酶对 SARS-CoV-2 感染的贡献外，抑制金属蛋白酶对于预防可能导致器官损伤的细胞死亡也很重要。我们的研究为 COVID-19 特有的复杂发病机制提供了新的见解，这与有效治疗方法的开发有关。