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. membrane fusion ͉ proteolytic processing ͉ virus entry E nveloped viruses access their host cells by a process of membrane fusion that is mediated by a specific fusion, or ''spike'' protein, encoded by the virus and embedded in the viral envelope (1, 2). Such proteins are currently grouped into 3 distinct structural classes, with the so-called class I fusion proteins typically primed for fusion activation by proteolytic cleavage (3, 4). Fusion activation can be activated by low pH, receptor binding, or a combination of the two, with the cleavage event typically occurring in the vicinity of the viral fusion peptide, which becomes exposed upon activation-dependent conformational changes of the spike protein and initiates the fusion reaction following its insertion into the host cell membrane (5). In several cases, such as highly pathogenic avian influenza virus, mutations in the cleavage site (monobasic vs. polybasic) and subsequent changes in the molecular basis of proteolytic cleavage by trypsin-like or furin-like enzymes have profound implications on virulence (6, 7). As such, understanding spike protein cleavage is fundamental to an understanding of viral pathogenesis.The severe acute respiratory syndrome coronavirus (SARSCoV) emerged in 2003 as a significant threat to human health, and coronaviruses still represent a leading source of novel viruses for emergence into the human population. The coronavirus spike protein (S) mediates both receptor binding (via the S1 domain) and membrane fusion (via the S2 domain) and shows many features of convention...

Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis

“…This site is analogous to the S2′ site in SARS-CoV S (Belouzard et al, 2009). The authors devised a clever experimental setup using conditional biontinylation assay that allows specific labeling and detection of MHV S proteins that have undergone the fusion process.…”

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin

“…Here, the cleavage events seem to occur sequentially since mitigating cleavage at the 544 RLHK 547 motif also blocks cleavage at the 518 RRRR 521 motif, but not vice versa. A similar sequential proteolytic cleavage of the fusion protein of the SARS (severe acute respiratory syndrome) coronavirus has been reported (Belouzard et al, 2009). It is postulated that cleavage at the first site allows subsequent cleavage at the second site, which in turn results in full fusion activity of the protein (Belouzard et al, 2012).…”

Molecular stability of a vaccine strain of Canine coronavirus after serial passages in A72 cells

“…Though the region of the S gene targeted in this study is responsible for membrane fusion and syncytial formation and has a higher mutation pressure than the S1 region, responsible for binding to cellular receptors (LAI; CAVANAGH, 1997), nucleotide polymorphism may also be found in S2 region (GALLAGHER; BUCHMEIER, 2001) allowing also virus entry into a variety of types of cells in trypsin-independent way (BELOUZARD et al, 2009;BORUCKI et al, 2013).…”