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Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.

ABSTRACT: The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1-S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2', R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell-cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2' cleavage site within S2 793-KPTKR-797 (S2'), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2' position allowed trypsin-independent cell-cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1-S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2'), acting in concert with the S1-S2 cleavage site to mediate membrane fusion and virus infectivity.

SUBMITTER: Belouzard S

PROVIDER: S-EPMC2660061 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Proteolytic activation of SARS-CoV-2 spike protein.

Project description:Spike (S) protein cleavage is a crucial step in coronavirus infection. In this review, this process is discussed, with particular focus on the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared with influenza virus and paramyxovirus membrane fusion proteins, the cleavage activation mechanism of coronavirus S protein is much more complex. The S protein has two cleavage sites (S1/S2 and S2'), and the cleavage motif for furin protease at the S1/S2 site that results from a unique four-amino acid insertion is one of the distinguishing features of SARS-CoV-2. The viral particle incorporates the S protein, which has already undergone S1/S2 cleavage by furin, and then undergoes further cleavage at the S2' site, mediated by the type II transmembrane serine protease transmembrane protease serine 2 (TMPRSS2), after binding to the receptor angiotensin-converting enzyme 2 (ACE2) to facilitate membrane fusion at the plasma membrane. In addition, SARS-CoV-2 can enter the cell by endocytosis and be proteolytically activated by cathepsin L, although this is not a major mode of SARS-CoV-2 infection. SARS-CoV-2 variants with enhanced infectivity have been emerging throughout the ongoing pandemic, and there is a close relationship between enhanced infectivity and changes in S protein cleavability. All four variants of concern carry the D614G mutation, which indirectly enhances S1/S2 cleavability by furin. The P681R mutation of the delta variant directly increases S1/S2 cleavability, enhancing membrane fusion and SARS-CoV-2 virulence. Changes in S protein cleavability can significantly impact viral infectivity, tissue tropism, and virulence. Understanding these mechanisms is critical to counteracting the coronavirus pandemic.

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Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

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