Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

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 have emerged that can evade the immunity in a host achieved through vaccination. 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 is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific 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 syncytia 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 endosome pathways could be an effective strategy by which to cure COVID-19 in the future.Author SummaryTo develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. 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. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This 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. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.