Proteolytic activation of the S1 / S2 site of the SARS-CoV-2 peak

Understanding the process of invasion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is crucial in therapeutic designs. Depending on the approach, it also sheds light on the evolution of the virus and its infection mechanism. In this study, the proteolytic activation of the spike protein of SARS-CoV-2, involved in the entry of the host cell, is evaluated in detail.

The SARS-CoV-2 virus that causes COVID-19 (coronavirus disease) has infected more than 35.65 million people and claimed more than 1 million lives worldwide. The rise in pathogenic strains of human coronavirus (CoV), with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, Middle East respiratory syndrome coronavirus (MERS-CoV) first emerged in 2012.

It is essential to understand the virus-host interaction and the sequential processes that result from an infection.

In a recent article published in bioRxiv* preprint server, Cornell University researchers study proteolytic activation of SARS-CoV-2 spike sites, re-evaluating furin cleavage. Their results demonstrate that pre-cleavage of S1 / S2 is essential for plasma membrane entry into Calu-3 cells (a model lung epithelial cell line), but not for endosomal entry into Vero E6 cells (a line cell culture model).

Predicted structural model of the S proteins of SARS-CoV and SARS-CoV-2. (Inset) Shown S1 / S2 site augmentation with conserved R and S residues (red ribbon) and the unique PRRA four amino acid insert for SARS-CoV-2 (blue ribbon). The P’s denote the position of that amino acid in the S1 / S2 cleavage site, with P1-P5 referring to amino acids before the cleavage site and P1 ‘referring to amino acids after the cleavage site.

The study reports that in addition to furin, other proteases are also responsible for the processing of SARS-CoV-2 S1 / S2.

The SARS-CoV-2 peak glycoprotein (S) is used to mediate viral entry into host cells. It is a large transmembrane protein that decorates the virus particle.

Protein S is known to be cleaved at the S1 / S2 or S2 ‘site. This is essential for the entry of the virus, which occurs in the cell plasma membrane or the endosomal membrane. It is known that furin can cleave the S1 / S2 site of the spike protein, expanding viral tropism to lung cells. Viral tropism is the ability of different strains or viral isolates to infect different types of cells or tissues, inducing virus production as a result of infection.

The results of the initial viral infection can vary widely depending on the site and route of entry, types of infected cells, local immune cell responses, and the virus or host species. In this study, the authors analyze the furin S1 / S2 site in related CoVs.

Based on the binding strength and accessibility of the motif in the furin-binding pocket, the authors analyzed the probability of cleavage of the S1 / S2 site by furin, using the prediction tools PiTou and ProP. Although both algorithms agree with each other to predict furin cleavage, due to their better sensitivity and specificity, they use the PiTou algorithm for more detailed analysis. The algorithm predicts that the SARS-CoV-2 S1 / S2 site can be cleaved by furin, while the other proposed lineage precursors cannot be cleaved by furin, this is confirmed experimentally. Furthermore, the SARS-CoV-2 S1 / S2 site has increased furin recognition compared to the other B-betaCoVs; the site is also not optimal for furin cleavage. The authors discuss in detail the origin and evolution of the virus and the related importance of the cleavage site. They urge exploring alternative explanations for the SARS-CoV-2 S1 / S2 site as a cattle furin insert.

In this study, the authors used viral pseudoparticles to assess the functional importance of the S1 / S2 site for SARS-CoV-2 entry. The core of the pseudoparticle is a murine leukemia virus (MLV), and the viral envelope protein is decorated to accurately recapitulate the entry steps of its native counterpart. These particles contain a luciferase reporter, which integrates into the host cell genome and produces quantifiable luciferase after successful infection. This study confirmed that the envelope protein is causing infections. They observed a higher infectivity of SARS-CoV compared to SARS-CoV-2; it comes from more protein S incorporated into the former.

The study suggests that S1 / S2 cleavage is essential for TMPRSS2-mediated plasma membrane entry, but not for cathepsin L-mediated endosomal entry, and that furin may not be the only protease responsible for the S1 / cleavage event. S2.

The claimed SARS-COV-2 S1 / S2 site is essential for the virus to enter the plasma membrane of respiratory cells; without cleavage, the virus would be endocytosed and would not be effective in infection. It is also known that the activation of this site appears to play a role in the immune response against SARS-CoV-2. The authors show that other proteases also need to be evaluated. The general observations of the study support that the role of the SARS-CoV-2 S1 / S2 site is to expand viral tropism to lung cells.

*Important news

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.