Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.
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ABSTRACT:
SUBMITTER: Simmons G
PROVIDER: S-EPMC3889862 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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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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Project description:Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.
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