ABSTRACT
The SARS-CoV-2 Omicron variant reportedly displays decreased usage of the cell surface entry pathway mediated by the host transmembrane protease, serine 2 (TMPRSS2) and increased usage of the endosomal entry pathway mediated by cathepsin B/L. These differences result in different cell tropisms and low fusogenicity from other SARS-CoV-2 variants. Recent studies have revealed that host metalloproteases are also involved in cell surface entry and fusogenic activity of SARS-CoV-2, independent of TMPRSS2. However, the involvement of metalloproteinase-mediated cell entry and fusogenicity in Omicron infections has not been investigated. Here, we report that Omicron infection is less sensitive to the metalloproteinase inhibitor marimastat, like the TMPRSS2 inhibitor nafamostat, and is more sensitive to the cathepsin B/L inhibitor E-64d than infections with wild-type SARS-CoV-2 and other variants. The findings indicate that Omicron preferentially utilizes the endosomal pathway rather than cell surface pathways for entry. Moreover, the Omicron variant also displays poor syncytia formation mediated by metalloproteinases, even when the S cleavage status mediated by fusion-like proteases is unchanged. Intriguingly, the pseudovirus assay showed that a single mutation, H655Y, of the Omicron spike (S) is responsible for the preferential entry pathway usage without affecting the S cleavage status. These findings suggest that the Omicron variant has altered entry properties and fusogenicity, probably through the H655Y mutation in its S protein, leading to modulations of tissue and cell tropism, and reduced pathogenicity. Author summaryRecent studies have suggested that the SARS-CoV-2 Omicron variant displays altered cell tropism and fusogenicity, in addition to immune escape. However, comprehensive analyses of the usage of viral entry pathways in Omicron variant have not been performed. Here, we used protease inhibitors to block each viral entry pathway mediated by the three host proteases (cathepsin B/L, TMPRSS2, and metalloproteinases) in various cell types. The results clearly indicated that Omicron exhibits enhanced cathepsin B/L-dependent endosome entry and reduced metalloproteinase-dependent and TMPRSS2-dependent cell surface entry. Furthermore, the H655Y mutation of Omicron S determines the relative usage of the three entry pathways without affecting S cleavage by the host furin-like proteases. Comparative data among SARS-CoV-2 variants, including Omicron, may clarify the biological and pathological phenotypes of Omicron but increase the understanding of disease progression in infections with other SARS-CoV-2 variants.
ABSTRACT
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.
ABSTRACT
We reported four cases of agricultural chemicals poisoning treated in our hospital during the past two years. These comprised two Paraquat poisoning and two Sumition poisoning cases. One Paraquat poisoned patient and one Sumition poisoned patient died of respiratory failure. The survivor of Paraquat poisoning had drank more Paraquat solutions than the dead case. However, the patient vomitted many times, and received medical treatment promptly, including gastric lavage, hemodialysis, and hemoperfusion.<BR>While on the other hand, it took a long time before starting of hemodialysis and hemoper-fusion on the dead case of Sumition poisoning. Because the poison was unknown when the patient was sent to our hospital. Our experience suggesuted that every time we examine a case of agricultural chemical poisoning, we have to treat a patient intensively from the beginning.
