A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Understanding how SARS-CoV-2 enters human cells is a high priority for deciphering its mystery and curbing its spread. A virus surface spike protein mediates SARS-CoV-2 entry into cells. To fulfill its function, SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases. Here we investigated receptor binding and protease activation of SARS-CoV-2 spike using biochemical and pseudovirus entry assays. Our findings have identified key cell entry mechanisms of SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD, supporting efficient cell entry. Second, paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 spike is comparable to or lower than that of SARS-CoV spike, suggesting that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is preactivated by proprotein convertase furin, reducing its dependence on target cell proteases for entry. The high hACE2 binding affinity of the RBD, furin preactivation of the spike, and hidden RBD in the spike potentially allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance. These features may contribute to the wide spread of the virus. Successful intervention strategies must target both the potency of SARS-CoV-2 and its evasiveness.
Discussion
With mounting infections, fatalities, and economic losses caused by SARS-CoV-2, it is imperative that we understand the cell entry mechanisms of SARS-CoV-2. However, recent studies have presented puzzling and sometimes conflicting findings on how SARS-CoV-2 enters cells, raising pressing scientific questions. For example, which virus binds to hACE2 more tightly, SARS-CoV-2 or SARS-CoV? What is the role of furin in SARS-CoV-2 entry? How does SARS-CoV-2 successfully evade human immune surveillance while maintaining its high cell infectivity? The current study addresses these questions by detailing the cell entry mechanisms of SARS-CoV-2.
Receptor recognition is an important determinant of coronavirus infection and pathogenesis. It is also one of the most important targets for host immune surveillance and human intervention strategies. The current study and other recent studies have revealed two patterns of results on the hACE2 binding affinity of SARS-CoV-2. First, with regard to the RBD, SARS-CoV-2 RBD has significantly higher hACE2 binding affinity than SARS-CoV RBD does. This was shown in our recent study using SPR assay as well as structural and mutagenesis analyses. In addition, using protein pull-down assay, the current study confirmed that SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD does. Second, despite the potency of its RBD’s binding to hACE2, the entire SARS-CoV-2 spike does not bind to hACE2 any more strongly than SARS-CoV spike does. Using protein pull-down assay, the current study showed that SARS-CoV-2 spike binds to hACE2 less strongly than SARS-CoV spike does. Another study using flow cytometry assay yielded similar results. A third study using Blitz assay showed that SARS-CoV-2 and SARS-CoV spikes have similar hACE2 binding affinities. Note that the hACE2 binding affinities of SARS-CoV RBD and SARS-CoV-2 spike should not be compared directly with each other. These findings therefore present a paradoxical pattern of results: Although SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD, its spike has hACE2 binding affinity comparable to or lower than SARS-CoV spike. These contrasting patterns between the RBD and the entire spike are particularly compelling in the current study because they were observed using the same method and under the same testing conditions. The dynamic state of the RBD in coronavirus spikes may explain this paradox. The RBD in coronaviruses can be in either a standing-up state, which enables receptor binding, or a lying-down state, which does not bind to the host receptors. Cryo-EM studies have shown that, in SARS-CoV spike, the RBD is mostly in the standing-up state; however, in SARS-CoV-2 spike, the RBD is mostly in the lying-down state. Therefore, compared to SARS-CoV, although SARS-CoV-2 RBD has higher hACE2 binding affinity, it is less accessible, resulting in comparable or lower hACE2 binding affinity for SARS-CoV-2 spike
To maintain its high infectivity while keeping its RBD less accessible, SARS-CoV-2 relies on a second strategy—host protease activation. Host protease activation is a significant determinant of coronavirus infection and pathogenesis, and a significant target for host immune surveillance and human intervention strategies. Using a combination of mutagenesis, protease inhibitors, and siRNA approaches, here we showed that furin preactivation enhances SARS-CoV-2 pseudovirus entry into different types of hACE2-expressing cell lines, including lung epithelial and lung fibroblast cell lines. We also showed that cell surface protease TMPRSS2 and lysosomal cathepsins activate SARS-CoV-2 pseudovirus entry and that both TMPRSS2 and cathepsins have cumulative effects with furin on SARS-CoV-2 entry. In comparison, SARS-CoV pseudovirus entry is activated by TMPRSS2 and cathepsins, but not furin. Furin preactivation allows SARS-CoV-2 to be less dependent on target cells, enhancing its entry into some target cells, particularly cells with relatively low expressions of TMPRSS2 and/or lysosomal cathepsins. This has also been observed with furin-preactivated avian influenza viruses. However, a recent study showed that furin preactivation enhances SARS-CoV-2 pseudovirus entry into BHK cells (baby hamster kidney fibroblast cells), but reduces SARS-CoV-2 pseudovirus entry into Vero cells (African green monkey kidney epithelial cells). These seemingly conflicting results can be explained by how coronavirus entry is regulated by proteases. Protease activation of coronavirus spikes potentially leads to the final structural change of coronavirus S2 needed for membrane fusion; this process is irreversible and needs to be tightly regulated. Indeed, it has been shown that, on SARS-CoV-2 virus particles, many spike molecules have already undergone the final structural change. Hence, in principle, virus particles preactivated by furin may have unchanged or reduced entry efficiency in some types of cells with high expressions of TMPRSS2 and/or lysosomal proteases; this may particularly be the case in vitro for virus particles that are not fresh, as the final conformational change of spike molecules may occur slowly spontaneously or be facilitated by environmental factors (e.g., high temperature, physical force, or some chemicals) . Overall, furin preactivation can facilitate SARS-CoV-2 to enter some types of cells (particularly those with low expressions of TMPRSS2 and/or lysosomal cathepsins)
Reference & Source information: https://www.pnas.org/
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