This post was reviewed by Dr. Sebastian Lequime, one of our subject matter experts.
This group of researchers from Germany investigated the multibasic amino acid sequence in the S1/S2 cleavage site on the spike glycoprotein of SARS-CoV-2. They wanted to know how important this sequence is for cleavage and entry. They also found that serine proteases (enzymes that cleave proteins) are involved in the spike protein cleavage. They identified that the human cellular serine protease, furin, cleaves the S1/S2 site and it is necessary for SARS-CoV-2 entry into lung cells. Their work also shows that furin cleaving the S1/S2 site is essential for allowing spike-protein mediated cell-to-cell fusion, which increases how well the virus travels from cell to cell. Primarily, this research suggests that the multibasic amino acid sequence in the S1/S2 site is essential for SARS-CoV-2 to infect human lung cells. With this new knowledge, some new therapeutic options may be investigated to combat COVID-19.
The paper we will be demystifying today can be found here, if you’d like to follow along.
This small group of researchers from the University of Gottingen and the Leibniz Institute for Primate research has previously shown that the SARS-CoV-2 spike glycoprotein requires a special type of enzyme called TMPRSS2 to infect human lung cells. TMPRSS2 is also known as serine protease and it is a cellular enzyme found in humans and other primates. I will explain what a serine protease is in a bit, but what these scientists predicted was that other cellular enzymes are also necessary for SARS-CoV-2 infection. They believe that these other enzymes act on the spike protein to help with SARS-CoV-2 entry into lung cells.
Before we dive into what it is these scientists discovered about the spike protein, let’s first remind ourselves how the spike protein facilitates the entry of the SARS-CoV-2 virus into our lung cells. As we remember, the spike protein is broken up into two subunits, the S1 and the S2 subunit. As we know the S1 subunit is made to bind to receptors on our cells and the S2 subunit is used to fuse the virus with the cell. For the S2 subunit to successfully fuse the virus to the cell, it has to use enzymes. The enzymes are involved in cleaving the spike protein at two special points on the protein itself. One enzyme will cleave the first site, the S1/S2 site. The second enzyme will cleave the S2’ site on the spike protein.
Now the question that these scientists began to ask themselves was: ‘What are the enzymes involved in cleaving these two sites on the spike protein?’ Remember, these enzymes are found in our and many other animals’ cells and they recognize and can cleave this virus’ spike protein. Well, as I mentioned earlier TMPRSS2 is a serine protease. A serine protease is essentially an enzyme that cleaves peptide bonds. This TMPRSS2 enzyme is known to recognize and cleave the S2’ site of the SARS-CoV-2 spike protein.
What we don’t know is which enzyme is involved in cleaving the S1/S2 site of the SARS-CoV-2 spike protein. Other scientists showed that the S1/S2 cleavage site has the exposed loop (when we’re talking about proteins, a ‘loop’ is a string of amino acids that don’t form a particular folding pattern and works to connect two parts of a protein). As you may remember, amino acids are the building block for proteins. Amino acid residues can also carry a charge on them, which makes them basic, acidic, or neutral. So, depending on what kind of amino acid sequence you have, they cause a protein to fold in a particular shape and orientation. In SARS-CoV-2 spike protein, the exposed loop contains a long line of basic amino acid residues. Interestingly, many other SARS related coronaviruses do not have this long line of basic amino acid residues.
Scientists wanted to look at this string of basic amino acids more closely. To do this, the scientists first had to create mutant SARS-CoV-2 spike proteins that have a change in the protein sequence at the S1/S2 cleavage site. They inserted the S1/S2 monobasic (meaning one basic) string of amino acid residues that are present in other coronaviruses into the SARS-CoV-2 spike protein. They also created mutants that either had arginines removed from or added to the S1/S2. Arginine is an amino acid residue with a basic charge to it. Finally, they took the SARS-CoV virus and introduced the S1/S2 site from the SARS-CoV-2 spike protein and CoV-RaTG13 (a bat coronavirus) into the SARS-CoV S1/S2 site respectively.
As a sidenote, I do want to mention that these mutant spike proteins (and only the spike protein, the whole virus was not cloned or mutated), were made using expression vectors and using another virus that doesn’t infect humans to present the spike protein on. Expression vectors are a scientist’s tool to only express one type of protein you want to study in a model cell type (like a bacteria or yeast cell). By doing this, scientists can study the effects of mutations and other things on a gene or on a protein. In this study when they introduced mutations into the spike protein, the scientists discovered that the mutations did not make the virus better at viral entry.
By doing this, they were able to observe the effects of viral entry. The normal (wild type) SARS-CoV-2 S1/S2 site cleaved normally. But the exchanged SARS-CoV-2 S1/S2 spike protein and the deleted arginine mutant didn’t get cleaved properly. The mutated SARS-CoV-2 spike protein with extra arginine was cleaved to the same level, which showed the scientists that there was no increase in the cleavability of the S1/S2 site with more arginines added.
What this means is that for efficient and high cleavability of the S1/S2 SARS-CoV-2 spike protein, a lot of arginine residues need to be there.
Now that they knew this, the scientists now wanted to figure out what the protease (remember this is a type of enzyme that cleaves proteins) required for processing and cleaving the S1/S2 spike protein site is. They noticed that the S1/S2 amino acid sequence fits the minimum requirement for the furin protease. This means that this human serine protease, known as furin, can cleave the S1/S2 spike protein site.
To test if furin is the protease used to cleave the S1/S2 spike, the scientists used a furin inhibitor- which would block the activity of the S1/S2 site cleavage. If the S1/S2 site is still cleaved in the presence of a furin inhibitor, it would mean that furin might not be the right serine protease. But what these scientists saw was that furin was effectively inhibited and that the S1/S2 site was not cleaved.
That’s pretty cool! They also looked at whether the cleavage of the S1/S2 site was necessary for SARS-CoV-2 to cause mammalian cell-to-cell fusion. This cell-to-cell fusion would essentially help SARS-CoV-2 infect more cells. But they saw that no fusion occurred when furin was inhibited.
Now knowing that furin cleaves the S1/S2 site and TMPRSS2 cleaves the S2’ site, the next step was to look at if the cleavage of S1/S2 is necessary for viral entry in human lungs. When the researchers purposely blocked the S1/S2 site from cleaving (for the wild type and mutant SARS-CoV-2 viruses), there was an abrogated entry into the TMPRSS2 expressing human lung cells. This means entry was somewhat avoided. They also noted that the mutations that optimized the cleaving site in the S1/S2 site for furin didn’t increase entry into the lung cells they tested.
However, when they looked at entry into cells that didn’t express TMPRSS2, blocking furin didn’t prevent entry (this was expected as it was previously seen in a previous study). Regardless, this shows us something important, which is that the basic amino acids in the S1/S2 site are essential for SARS-CoV-2 entry into human lung cells. This also indicates that the S1/S2 site may need to be cleaved first to activate the TMPRSS2 protease which is needed to cleave the S2’ site. This order of cleavage and activation of serine proteases is needed for viral entry into lung cells.
They suggest that because there are known inhibitors for furin and TMPRSS2, we could potentially use them as a treatment option for COVID-19. But of course, much more clinical research must be done before we can consider these as treatment options.