Tmprss2

The spike glycoprotein of SARS-CoV-2 contains a cleavage site for host cell proteases called furins. Deciphering the role of this cleavage site during infection is important for understanding the origin of the pandemic virus and its disease pattern in humans.

Back in February it was not known if the furin site in the SARS-CoV-2 was cleaved by cell proteases, and whether its presence is required for infectivity. Both questions have now been answered.

The figure shows amino acids at cleavage sites in the spike glycoproteins of various CoVs. A furin site is present in the spike glycoprotein of HCoV-OC43, HCoV-HKU1, MERS-CoV and SARS-CoV 2. It is called a multibasic site because it contains multiple basic (arginine) amino acids. The spike glycoproteins of HCoV-NL63, HCoV-229E, and SARS-CoV do not contain this multibasic cleavage site. Neither do SARS-related CoVs found in bats, including RaTG13, the virus with the closest overall genome sequence identity with SARS-CoV-2.

To study cleavage and function of the furin site, various CoV spike glycoproteins were engineered into vesicular stomatitis virus particles. This manipulation allowed the study of infected cells without the need for a BSL3 facility. The spike glycoprotein of SARS-CoV-2 was efficiently cleaved, while that of SARS-CoV or RaTG13 was not. Furthermore, when the SARS-CoV-2 furin site was exchanged with the corresponding sequence from SARS-CoV or RaTG13, no cleavage was observed. That cleavage was mediated by furins was verified by using specific protease inhibitors.

Cleavage of CoV spike glycoproteins is required for fusion of the viral and cell membranes upon entry. VSV harboring the spike of SARS-CoV-2 caused fusion of a human lung cell line; substitution of the furin cleavage site with the corresponding sequence from SARS-CoV or RaTG13 prevented cell fusion. However, VSV harboring the spike of SARS-CoV did cause fusion of these lung cells, due to cleavage by a different protease. These observations demonstrate that the furin cleavage site in the spike glycoprotein is essential for entry of SARS-CoV-2 into lung cells. In contrast, a monobasic cleavage site is sufficient for entry of SARS-CoV.

The activation of the spike glycoproteins of SARS-CoV-2 and MERS-CoV are therefore similar. They both must first be cleaved by furins followed by cleavage by a different cell protease, TMPRSS2.

An interesting question is the origin of the furin cleavage site it SARS-CoV-2. Its closest relative, the bat isolate RaTG13, does not have this site. Nor do any of the other bat SARS-like CoVs or the pangolin CoVs that have been isolated. However recently a newly isolated bat SARS-like CoV, RmYN02, was shown to contain a poly basic amino acid insertion in the spike glycoprotein. This observation supports the hypothesis that the furin cleavage site in SARS-CoV-2 arose by recombination among bat viruses in nature.