Determining the Cell Entry Mechanism of SARS-CoV-2

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November 12, 2021

The emergence of SARS-CoV-2 has caused much damage throughout the world. Deaths, lockdowns, and economic stagnation have all been a result of the virus. Due to its deleterious effects, much research has been done to figure out the mechanism in which SARS-CoV-2 enters cells. Determining how the virus enters cells will assist in ultimately developing strategies to curb its transmission.

To gain insight into how SARS-CoV-2 enters cells, many have looked to SARS-CoV, mainly to determine if SARS-CoV treatment strategies can be applied to treat SARS-CoV-2. There are similarities between SARS-CoV and SARS-CoV-2, including:

A trimer S1 subunit
Human angiotensin-converting enzyme (hACE2) as the target cell receptor that binds the spike protein’s receptor-binding domain (RBD)
Proteases that cleave the S1/S2 junction of the spike protein

SARS-CoV and SARS-CoV-2 also exhibit some differences, namely with respect to target cell binding efficacy, target cell binding affinity, types of proteases for cleaving the S1/S2 junction, and antibody binding.

Cell binding efficacy

Much of the research currently being carried out involves determining the binding efficacy of the SARS-CoV-2 RBD to a target cell’s surface. Regarding SARS-CoV, its spike protein has three receptor-binding S1 heads, comprising the S1 subunit, on top a trimeric membrane fusion S2 stalk, comprising the S2 subunit. Previous papers have found that the SARS-CoV spike protein binds to hACE2 via SARS-CoV RBD, which is connected to the S1 subunit, with the ultimate result being target cell entry via endosomes and viral membranes fusing to target cell lysosomal membranes (Shang et al., Li). A SARS-CoV RBD, attaching to a target cell hACE2 receptor, switches from an up to a down configuration, the up configuration providing more potent binding than the down position. Shang et al. contend that SARS-CoV-2 spike protein RBD is mainly in the down position, indicating predominantly poor binding to the target cell.

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Like SARS-CoV, SARS-CoV-2 spike protein also has a S1 trimer subunit, with Wrapp et al. finding that the trimer has a RBD that is predominantly in the up position, indicating strong receptor binding. SARS-CoV-2 was also found to bind hACE2, binding even more strongly than SARS-CoV.

Cell binding affinity

Shang et al. found that hACE2 binding affinity of the SARS-CoV-2 spike protein is either similar to or lower than that of SARS-CoV spike protein. This suggests that, although SARS-CoV-2 RBD binding to hACE2 is strong, it is less available for binding to the target cell than the RBD of SARS-CoV.

Wrapp et al., however, found that SARS-CoV-2 RBD has a greater affinity of hACE2 than SAR-CoV RBD. They identified a 3.5-angstrom-resolution structure of the SARS-CoV-2 spike protein by cryo–electron microscopy and, using biophysical assays, observed that SARS-CoV-2 has as high as 10–20 times greater affinity for hACE2 than SARS-CoV, which they suggest may explain why SARS-Cov-2 spreads rather easily.

Protease activation

For the SARS-CoV viral membrane and target cell lysosomal membrane to fuse, the SARS-CoV spike protein is activated at the S1/S2 junction. Proteases TMPRSS2 and cathepsins facilitate this activation.

Hoffman et al. found that the spread of SARS-CoV-2 also depends on TMPRSS2 activity, speculating that furin-mediated cleavage at the S1/S2 junction of the spike protein might promote subsequent TMPRSS2-dependent entry into target cells. Shang et al. concur, stating that furin, a proprotein convertase that reduces the SARS-CoV-2 spike protein’s dependence on a target cell’s own proteases for cell entry, is a preactivator of cell entry, acting at the S1/S2 junction. This observation refutes previous research that asserts that furin cleavage at the S1/S2 junction does not lead to SARS-CoV-2 cell entry (Walls et al.). They do recognize that a furin cleavage site exists for the SARS-CoV-2 spike protein that does not exist for SARS-CoV, though.

As a means of inhibiting virus cell entry, serine protease inhibitor camostat mesylate, which blocks TMPRSS2 activity (Kawase et al., 2012, Zhou et al., 2015), has been approved in Japan for human use, but for an unrelated indication. This compound or related ones could potentially be used as an off-label treatment for SARS-CoV-2.

Antibody binding

Antibody binding to neutralize SARS-CoV-2 is a potential strategy to lessen the deleterious effects of SARS-CoV-2 spread. Wrapp et al. found that SARS-CoV RBD-specific monoclonal antibodies (S230, m396, and 80R), when tested for their binding efficiency to SARS-CoV-2, do not have appreciable binding to SARS-CoV-2, suggesting poor antibody cross-reactivity. Similarly, Ou et al. found that the binding efficiency of antibody T62 to SARS-CoV was appreciable, but was non-existent for SARS-CoV-2. The switching nature of the SARS-CoV-2 RBD from up to down configurations does not help matters, as antibody binding in the down configuration will be limited.

It is crucial to develop drug therapies that incorporate an antibody whose binding efficiency with SARS-CoV-2 RBD is greater than RBD binding with target cell hACE2. Recent work showed that recombinant ACE2 can inhibit SARS-CoV-2 infection in engineered human tissues (Monteil et al.), which suggests that blocking RBD from binding to target cell receptors is possible. Thus, an antibody drug with significantly higher RBD binding affinity than ACE2 can dominate over cell surface hACE2 in latching onto the RBD, blocking viral attachment.

Hoffman et al. found that there are some antibodies that neutralize both SARS-CoV and SARS-CoV-2, with varying results. Sera from three SARS patients decreased SARS-CoV-2 target cell entry, albeit not as well as SARS-CoV-driven cell entry. Rabbit sera raised against the S1 subunit of SARS-CoV decreased both SARS-CoV and SARS-CoV-2 driven cell entry.

In addition to antibodies that bind to the SARS-CoV-2 RBD, other strategies may decrease SARS-CoV-2 spread. One such strategy is developing vaccines and drugs that target the S2 subunit. Because the S2 subunit is less immunogenic than the RBD, though, such a strategy may have limited success. Another potential strategy to prohibit SARS-CoV-2 cell entry is targeting protease activators. However, because cell entry is mediated by more than one protease (furin, TMPRSS2), there would likely need to be multiple inhibitors to target all the proteases.

Determining that hACE2 is a receptor for the RBD of the SARS-CoV-2 spike protein, in addition to identifying proteases that are involved with its transmission into target cells (furin, TMPRSS2) are important in better understanding the SARS-CoV-2 cell entry mechanism. Ultimately, though, more work needs to be done, building upon this information, to develop strategies to suppress the virus’s spread.