A critical step in the race to develop treatments for COVID-19 is for scientists to gain a clear understanding of exactly how the virus enters our cells. This insight will support development of targeted anitviral treatments focused on blocking that pathway.

Research on the first SARS-CoV virus, which emerged in 2002 causing an epidemic, as well as on SARS-CoV-2, the related coronavirus that is now causing COVID-19, shows that in both cases a spike (S) protein that protrudes from the viral membrane binds to at least one protein, angiotensin-converting enzyme 2 (ACE2), on the surface of human cells. After binding, proteases, which are human enzymes that clip other proteins into pieces, cut, or “prime”, the spike protein to remove its outer segment, named S1, and reveal the inner segment, named S2. The spike protein S2 segment then causes fusion of the viral membrane with the human cell membranes, letting the viral genetic material enter the cell and start replicating. A recent post summarized this process highlighting the role of ACE2. In this post, I’ll go into more detail about the role that human proteases play in assisting the virus in entering our cells and highlight antiviral treatments targeting that interaction.

The SARS-CoV-2 spike protein: A tale of two segments

The SARS-CoV-2 spike protein is shaped somewhat like a screw with a larger head and a longer, thinner stalk (Figure 1). Three spike proteins bind to each other to form a trimer, which is shaped, predictably, like a bigger screw. The stalk is inserted into the viral membrane and holds the head outwards away from the virus. The larger head region and part of the stalk are called the S1 region of the spike protein. The remaining part of the stalk that’s closer to the viral membrane is called the S2 region.

Once it enters the body and comes into contact with respiratory system, gastrointestinal tract, blood vessel or other cells that express ACE2 on their surfaces, the spike protein’s S1 region binds to ACE2 on the cell surface and tethers the virus to the outside of the human cell. This is the first step in the viral replication process.

SARS-CoV-2 enters cells one way or another

Once the virus has bound itself to the cell, it has two different potential pathways for entry (Figure 2). Which pathway is used depends on whether or not human proteases are present to “prime” the spike protein. The presence of proteases depends on the type of human cell that the virus is entering and on the particular conditions at that cell. Several human proteases can cleave the spike protein, including transmembrane serine proteinase 2 (TMPRSS2), furin, elastase and trypsin. TMPRSS2 is expressed by human lung cells. Thus, it is thought that it plays an important part in virus entry into respiratory system cells.

If these proteases happen to be present near the spike-ACE2 binding interface, they will cleave the spike protein to expose the S2 region, and specifically the fusion peptide region, of the spike protein. This fusion peptide region of spike is made of more hydrophobic, or lipid-like, amino acids, and it inserts into the lipid-containing cell membrane to induce viral membrane ̶ cell membrane fusion and subsequent entry of the viral genome into the cell (Figure 2a). This cleavage must occur after spike-ACE2 binding. If it occurs before, the virus is less able to infect the cell.

If proteases don’t happen to be present near the spike ̶ ACE2 binding interface, the virus will enter the cell by a different pathway called endocytosis (Figure 2b). In this process, the coronaviruses bound to ACE2 proteins outside the cell are engulfed by an indentation in a small region of the cell’s membrane, which then pinches off to form an endocytic vesicle that brings the outside material into the cell. After this happens, the endocytic vesicle fuses with an intracellular membrane-walled vesicle called an endosome. In the endosome, there are proteases present, including one called cathepsin L, that can cleave the spike protein and expose its fusion peptide region. The fusion peptide then mediates fusion of the viral membrane with the endosome’s membrane and thereby induces subsequent entry of the viral genome into the cell.

Recent evidence suggests that there may be a third way that SARS-CoV-2 can enter cells. When the virus is replicating and making new virus particles inside cells, some of the spike proteins might be pre-cleaved, or pre-primed, by a human protease called furin during the new virus assembly process. This means that once the virus breaks out of the cell, those viruses with pre-primed spike proteins can fuse with and infect other cells even if those other cells have low levels of proteases present for one of the two “normal” spike protein cleavage pathways described above.

Planning a counter-attack

Researchers are working hard on drugs that can target the spike ̶ ACE2 ̶ membrane fusion ̶ endocytosis part of the infection lifecycle to hinder COVID-19. Our previous post highlighted recombinant soluble ACE2 as a potential treatment. It works by inactivating the spike protein before SARS-CoV-2 can bind to ACE2 on the surface of cells. However, many other drug candidates are under consideration as well.

Nafomastat and MI-1851 inhibit the proteases involved in spike protein cleavage, TMPRSS2 and furin, respectively, showing potential to reduce SARS-CoV-2 infection in the test tube. Peptides, which are very short proteins that are similar to small regions of the spike protein, have been shown to inhibit fusion of the viral and human cell membranes by “clogging up” the primed spike protein on the virus as it’s changing shape during the membrane fusion process. This prevents viral entry. Finally, PIKfyve inhibitors are known blockers of SARS-CoV-2 infection. PIKfyve is a human lipid kinase, which is an enzyme that adds a phosphate group to specific lipids. As PIKfyve is involved in endosomal metabolism in the endocytic pathway of viral entry, PIKfyve inhibitors have antiviral activity.

These are just a few of the many drug candidates being studied as SARS-CoV-2 viral entry inhibitors. However, there are many targets available to researchers seeking treatments for COVID-19. The spike protein, ACE2, the proteases that cleave the spike protein and components of the endocytic pathway are all possibilities being studied, and there are many substances that have antiviral activity relative to each of these targets. To help scientists identify some of those potential candidates faster, CAS has released an open source dataset assembled from CAS REGISTRY^® that includes known anti-viral drugs and related chemical compounds that are structurally similar to known antivirals. Learn more and download it and other CAS open access COVID-19 resources here.