A study conducted by researchers at the University of Kentucky and the Washington University School of Medicine in the USA has revealed that mutations in the proteolytic cleavage site within the S2 subunit can alter the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, affecting viral entry into host cells. The study’s findings are currently available on the bioRxiv* preprint server.
Since its emergence in December 2019, SARS-CoV-2, the causative pathogen of coronavirus disease 2019 (COVID-19), has already infected over 100 million people and claimed over 2.145 million lives globally. SARS-CoV-2 is known to primarily spread via respiratory droplets, and the interaction between viral spike protein and the host angiotensin-converting enzyme 2 (ACE2) is a prerequisite for viral entry into host cells. Of two distinct subunits of SARS-CoV-2 spike protein, S1 is required for ACE2 binding, and S2 is required for viral-host cell membrane fusion, which is the major step for viral entry. For the priming of spike protein and initiation of cell-cell fusion, two proteolytic cleavage processes, one occurring at the border of S1/S2 subunits and another one occurring within the S2 subunit, are indispensably important.
In the current study, the scientists investigated the effects of mutations in the viral spike protein on protein stability, proteolytic cleavage events, and cell-cell fusion.
They have conducted a series of experiments in a variety of mammalian and bat cell lines and observed that the proteolytic cleavage of spike protein readily occurs at the border of S1/S2 subunits and that furin is the primary protease to catalyze this proteolytic cleavage. Moreover, they have observed that furin when present in the spike-expressing effector cells increases the cell-cell fusion, whereas TMPRSS2, another cellular protease required for spike priming, increases the cell-cell fusion when present in ACE2-expressing target cells. Interestingly, they have noticed that a high level of cell-cell fusion can be achieved in effector cells with high endogenous levels of TMPRSS2. As suggested by the scientists, TMPRSS2-mediated increase in cell-cell fusion might be due to its action on both spike protein and ACE2 receptor.
Besides cellular proteases, they have also investigated the effect of neuropilin-1, which is a co-receptor for viral entry and may be responsible for SARS-CoV-2 entry into the neuronal network. However, they could not observe any alteration in cell-cell fusion when neuropilin-1 is co-expressed with ACE2 in target cells or with spike protein in effector cells, indicating that neuropilin-1 is not essential for cell-cell fusion and viral entry.
Using both mammalian and bat cell lines, they have observed that the canonical furin cleavage motif at the S1/S2 border of SARS-CoV-2 spike protein differs from that present in other similar coronaviruses. Interestingly, the analysis of wild-type spike protein in bat cells has revealed that furin can recognize and cleave this motif at a slower rate. While some level of cleavage has been observed in bat cells expressing spike protein mutated at the furin cleavage motif, no such cleavage has been observed in monkey and human cell lines. These observations suggest that certain mutations in the bat coronaviruses facilitate the cleavage of canonical motifs by human proteases, which might be important for the zoonotic transmission of coronaviruses.
Importantly, they have observed that mutations in the proteolytic cleavage motifs at the S1/S2 border or within the S2 domain can reduce the initial cleavage at the S1/S2 border during the synthesis of viral protein. This indicates that an alteration in protein conformation is possible due to mutations that occur downstream of the S1/S2 border. Moreover, they have identified that mutations in two specific residues at the S2 subunit can alter the protein fusion function without altering protein expression and cleavage. Of these two residues, one is present in the internal fusion peptide, and another one is present in the cytoplasmic tail. These findings indicate the importance of these regions in the protein fusion process.
Interestingly, the researchers observed that D614G mutation, which is a predominant spike mutation causing increased viral infectivity, does not alter protein stability, cleavage, and cell-cell fusion process. Specifically, they have observed that S1 and S2 domain mutations observed in currently circulating SARS-CoV-2 strains do not alter the turnover of the spike protein.
The study provides valuable insight into SARS-CoV-2 spike protein stability, proteolytic processing, and cell-cell fusion, which are important for understanding the viral transmission process and identifying novel therapeutic targets.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.