June 23, 2022

Study Confirms Omicron Variant Shifted to Better Utilization of Cathepsin B/L Entry Pathway

The evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has led to the emergence of many new variants. These variants are different from the original strain of SARS-CoV-2, which was first reported in China in 2019. Some of the variants that emerged have been classified as variants of concern (VOC), as they are able to evade the immune response induced after infection or immunization with COVID-19.

To study: Evidence for increased cathepsin B/L and decreased use of TMPRSS2 for cell entry by SARS-CoV-2 variant Omicron. Image Credit: MedMoMedia/Shutterstock


The new SARS-CoV-2 Omicron variant has been classified as a COV and has become the dominant strain circulating in many countries. It contains thirty-seven mutations in the spike (S) protein, compared to the original strain of SARS-CoV-2, and can evade immune protection generated by vaccination or natural infection. Previous studies have shown that the original strain of SARS-CoV-2 exhibited broad cellular tropism.

Recent studies have shown that due to a high number of mutations, Omicron exhibits altered cellular tropism and entry mode compared to other SARS-CoV-2 variants. For example, SARS-CoV-2 pseudotyped virus containing variant B.1 or Delta spike protein showed higher entry efficiency than Omicron pseudotyped virus, in Caco-2 (human colon cells) and Calu- 3 (human lung cells). ) cells.

However, this is not the case in other cell types. For example, the Omicron pseudotyped virus can invade Vero (African green monkey kidney cells) and 293T (human kidney cells) cells more efficiently than other variants. Similar results have been reported by studies that tested for infection with live SARS-CoV-2 virus. These studies revealed that, in Calu-3 cells, infection with the Delta variant spread much faster than the Omicron variant.

A new study

A new study published on the bioRxiv* The preprint server analyzed new data on the Omicron variant and indicated the altered cellular entry pathways compared to other variants. This study used a mathematical model to quantify the extent of different use of the input pathways modified by the Omicron variant.

Previous studies have shown that the Omicron spike protein binds strongly to soluble human ACE2. Researchers observed that reducing spike protein density decreased Omicron’s entry efficiency. They explained that after SARS-CoV-2 binds to host ACE2, successful entry requires cleavage of the spike protein into two subunits by host proteases by a serine transmembrane protease (TMPRSS2) or by the cysteine ​​proteases Cathepsin B and Cathepsin L (cathepsin B/L) in endosomal vesicles.

Previous studies have indicated that the two proteases work independently to allow entry of SARS-CoV-2 into host cells, following two different pathways. In this new study, researchers assessed infection by the SARS-CoV-2 pseudotyped virus of multiple cell lines using a mathematical model of SARS-CoV-2 entry.

They reported differential use of TMPRSS2 and Cathepsin B/L entry pathways, between the original SARS-CoV-2 strain and the Omicron variant, in all cell lines. For example, Vero cells have extensively utilized the cathepsin B/L pathway for virus entry, while Calu-3 cells allow entry through the TMPRSS2 pathway. The original strain of SARS-CoV-2 showed the ability to use both pathways. The preferred pathways could be related to the level of expression of the two proteases.

Principle results

The authors of this study hypothesized that the Omicron variant may use the TMPRSS2 and Cathepsin B/L entry pathways differently than the original or other SARS-CoV-2 variants, which could lead to its tropism differential cell. They tested this theory and observed a correlation between the entry efficiency of the Omicron virus and the relative use of the two entry routes by the parent strain.

The scientists reported that the original strain primarily used the TMPRSS2 entry pathway to enter both Calu-3 and Caco-2 cells. However, among viral variants, the efficiency of entry into these cell lines using the TMPRSS2 entry pathway was much lower in the Omicron pseudotyped virus than in the B.1 and delta strains. Scientists revealed that Omicron cells more efficiently enter cells where use of the cathepsin B/L entry pathway by the original strain was dominant.

These results imply that compared to other SARS-CoV-2 variants, entry of the Omicron variant is relatively less efficient via the TMPRSS2 pathway and more efficient via the cathepsin B/L pathway. This observation is consistent with another study which reported that camostat mesylate, an inhibitor of TMPRSS2, was less effective in blocking the live Omicron variant than the Delta variant in VeroE6 cells. In contrast, cathepsin B/L inhibitors were more effective against the Omicron variant. The mathematical model provided the synergy between TMPRSS2 and cathepsin B/L inhibitors against SARS-CoV-2 infection.


The authors revealed that the altered cell tropism associated with the Omicron variant is linked to increased efficiency of Cathepsin B/L utilization and reduced efficiency of TMPRSS2 utilization. This study revealed that the increased average sensitivity of the cell population to virus entry is related to the use of both pathways. The scientists said that since the preference of the Omicron variant for the TMPRSS2 pathway is reduced and not entirely eliminated, combination therapy with TMPRSS2 and cathepsin B/L inhibitors might be a better treatment strategy.

*Important Notice

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

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