By Michael Greenwood, M.Sc.May 6 2021

It is well known that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds with the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface. This process facilitates host-virus membrane fusion, with cell entry achieved by host proteases.

The particular host protease utilized varies amongst betacoronaviruses, though SARS-CoV-2 appears to use cathepsins primarily, usually found in the late endosome, suggesting that SARS-CoV-2 enters the cell by the endocytotic pathway. MERS-CoV, by comparison, is thought to be activated predominantly by TMPRSS2, allowing entry directly at the cell surface, though SARS-CoV-2 has also been observed to use this mechanism.

It is possible that multiple triggers are required to allow entry, or that multiple routes are equally possible depending on cell type, but studies that utilize targeted proteolytic inhibitors to investigate these mechanisms may disturb ordinary membrane composition and function.

In a new study recently released on the bioRxiv* preprint server, a team of researchers from the University of Virginia, USA, used single-virus fusion experiments with exogenously controlled proteases to investigate the mechanism of activation directly. They found that the subcellular location of entry depends on the protease activity of the various components, and parallel pathways are possible.

How was the study performed?

The group immobilized a host cell plasma membrane expressing ACE2 and low levels of TMPRSS2 in a microfluidic flow cell, and labeled the virus with a lipid-phase fluorophore. Proteases were then added to the flow cell, and where they facilitated cell entry, the fluorophores became dequenched to highlight the subcellular location. Vero cell membranes were used for this purpose, and minimal fusion was observed between these cells and the pseudoviruses employed in the absence of exogenous protease.

For comparison, Calu-3 cells, which express higher levels of TMPRSS2 and similar levels of ACE2, could be more easily entered by the pseudoviruses. The pseudoviruses consisted of a viral membrane sourced from either HIV, Indiana vesiculovirus (VSV), or murine leukemia viruses (MLV) expressing the SARS-CoV-2 spike protein. The group notes that the VSV membrane was able to support a greater density of the spike protein, and thus entered the cell more quickly than the others.

The group tested four exogenously introduced proteases in combination with the pseudoviruses: TMPRSS11D, also known as human airway trypsin-like (HAT) protease, a serine protease found in airway tissues; Cathepsins B and L, late endosomal proteases; and TMPRSS2, the cell surface protease found on alveolar cells. Each of these proteases was able to activate the SARS-CoV-2 spike protein for fusion, and they each showed similar fusion times.

Based on this, the group suggests that proteolytic activation can occur at any point, prior to or following bonding with ACE2: in the extracellular medium by HAT, at the cell surface by TMPRSS2, or within an endosome by the Cathepsins. One route of cell entry is likely to be favored in tissues or cells with a greater concentration of any of the particular proteases studied here.

Protease inhibitors are well-explored as drug leads against SARS-CoV-2. While often demonstrating impressive virus inhibition in vitro, often fall short at the in vivo stage, to which the authors attribute these multiple opportunistic pathways.

*Important Notice

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.