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

TLDR

The SARS‑CoV spike protein contains an S1 receptor‑binding domain and an S2 fusion domain, and although cleavage at the S1‑S2 boundary and cathepsin L activity are known to influence entry, the precise proteolytic steps that trigger membrane fusion remain unclear. The study aimed to identify the specific proteolytic cleavage site within the S2 domain that activates SARS‑CoV fusion. The authors engineered furin cleavage sites at the S2′ position (R797) and at the S1‑S2 junction to assess their role in protease‑independent fusion. Mutation of R797 blocked trypsin‑dependent fusion, while the engineered furin sites enabled trypsin‑independent fusion that was markedly enhanced by the additional S1‑S2 site, indicating that cleavage at both positions cooperatively primes the spike for membrane fusion and viral infectivity.

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.